Method for promoting bone growth using activin-actriia antagonists

ABSTRACT

In certain aspects, the present invention provides compositions and methods for promoting bone growth and increasing bone density.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/849,313, filed Sep. 9, 2015, which is a continuation of U.S.application Ser. No. 14/027,542 (now U.S. Pat. No. 9,163,075), filedSep. 16, 2013, which is a continuation of U.S. application Ser. No.13/176,718 now U.S. Pat. No. 8,629,109), filed Jul. 5, 2011, which is acontinuation of U.S. application Ser. No. 12/284,862 (now U.S. Pat. No.8,067,360), filed Sep. 24, 2008, which is a continuation of U.S.application Ser. No. 11/603,485 (now U.S. Pat. No. 7,612,041), filedNov. 22, 2006, which claims the benefit of U.S. Provisional ApplicationNos. 60/739,462, filed Nov. 23, 2005, 60/783,322, filed Mar. 17, 2006,and 60/844,855, filed Sep. 15, 2006. The specifications of each of theforegoing applications are incorporated herein by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Aug. 1, 2018, is named1848179-019-107_Seq.txt and is 22,393 bytes in size.

BACKGROUND OF THE INVENTION

Disorders of the bone, ranging from osteoporosis to fractures, representa set of pathological states for which there are few effectivepharmaceutical agents. Treatment instead focuses on physical andbehavioral interventions, including immobilization, exercise and changesin diet. It would be beneficial to have therapeutic agents that promotebone growth and increase bone density for the purpose of treating avariety of bone disorders.

Bone growth and mineralization are dependent on the activities of twocell types, osteoclasts and osteoblasts, although chondrocytes and cellsof the vasculature also participate in critical aspects of theseprocesses. Developmentally, bone formation occurs through twomechanisms, endochondral ossification and intramembranous ossification,with the former responsible for longitudinal bone formation and thelater responsible for the formation of topologically flat bones, such asthe bones of the skull. Endochondral ossification requires thesequential formation and degradation of cartilaginous structures in thegrowth plates that serve as templates for the formation of osteoblasts,osteoclasts, the vasculature and subsequent mineralization. Duringintramembranous ossification, bone is formed directly in the connectivetissues. Both processes require the infiltration of osteoblasts andsubsequent matrix deposition.

Fractures and other structural disruptions of bone are healed through aprocess that, at least superficially, resembles the sequence ofdevelopmental events of osteogenesis, including the formation ofcartilaginous tissue and subsequent mineralization. The process offracture healing can occur in two ways. Direct or primary bone healingoccurs without callus formation Indirect or secondary bone healingoccurs with a callus precursor stage. Primary healing of fracturesinvolves the reformation of mechanical continuity across a closely-setdisruption. Under suitable conditions, bone-resorbing cells surroundingthe disruption show a tunnelling resorptive response and establishpathways for the penetration of blood vessels and subsequent healing.Secondary healing of bones follows a process of inflammation, softcallus formation, callus mineralisation and callus remodelling. In theinflammation stage, haematoma and haemorrhage formation results from thedisruption of periosteal and endosteal blood vessels at the site ofinjury. Inflammatory cells invade the area. In soft callus formationstage, the cells produce new vessels, fibroblasts, intracellularmaterial and supporting cells, forming granulation tissue in the spacebetween the fracture fragments. Clinical union across the disruption isestablished by fibrous or cartilaginous tissue (soft callus).Osteoblasts are formed and mediate the mineralization of soft callus,which is then replaced by lamellar bone and subjected to the normalremodeling processes.

In addition to fractures and other physical disruptions of bonestructure, loss of bone mineral content and bone mass can be caused by awide variety of conditions and may result in significant medicalproblems. Changes to bone mass occur in a relatively predictable wayover the life of an individual. Up to about age 30, bones of both menand women grow to maximal mass through linear growth of the endochondralgrowth plates and radial growth. After about age 30 (for trabecularbone, e.g., flat bones such as the vertebrae and pelvis) and age 40 (forcortical bone, e.g., long bones found in the limbs), slow bone lossoccurs in both men and women. In women, a final phase of substantialbone loss also occurs, probably due to postmenopausal estrogendeficiencies. During this phase, women may lose an additional 10% ofbone mass from the cortical bone and 25% from the trabecularcompartment. Whether progressive bone loss results in a pathologicalcondition such as osteoporosis depends largely on the initial bone massof the individual and whether there are exacerbating conditions.

Bone loss is sometimes characterized as an imbalance in the normal boneremodeling process. Healthy bone is constantly subject to remodeling.Remodeling begins with resorption of bone by osteoclasts. The resorbedbone is then replaced by new bone tissue, which is characterized bycollagen formation by osteoblasts, and subsequent calcification. Inhealthy individuals the rates of resorption and formation are balanced.Osteoporosis is a chronic, progressive condition, marked by a shifttowards resorption, resulting in an overall decrease in bone mass andbone mineralization. Osteoporosis in humans is preceded by clinicalosteopenia (bone mineral density that is greater than one standarddeviation but less than 2.5 standard deviations below the mean value foryoung adult bone). Worldwide, approximately 75 million people are atrisk for osteoporosis.

Thus, methods for controlling the balance between osteoclast andosteoblast activity can be useful for promoting the healing of fracturesand other damage to bone as well as the treatment of disorders, such asosteoporosis, associated with loss of bone mass and bone mineralization.

With respect to osteoporosis, estrogen, calcitonin, osteocalcin withvitamin K, or high doses of dietary calcium are all used as therapeuticinterventions. Other therapeutic approaches to osteoporosis includebisphosphonates, parathyroid hormone, calcimimetics, statins, anabolicsteroids, lanthanum and strontium salts, and sodium fluoride. Suchtherapeutics, however, are often associated with undesirable sideeffects.

Thus, it is an object of the present disclosure to provide compositionsand methods for promoting bone growth and mineralization.

SUMMARY OF THE INVENTION

In part, the disclosure demonstrates that molecules having activin orActRIIa antagonist activity (“activin antagonists” and “ActRIIaantagonists”) can be used to increase bone density, promote bone growth,and/or increase bone strength in particular, the disclosure demonstratesthat a soluble form of ActRIIa acts as an inhibitor of activin-ActRIIasignaling and promotes increased bone density, bone growth, and bonestrength in vivo. While most pharmaceutical agents that promote bonegrowth or inhibit bone loss act as either anti-catabolic agents (alsocommonly referred to as “catabolic agents”) (e.g., bisphosphonates) oranabolic agents (e.g., parathyroid hormone, PTH, when appropriatelydosed), the soluble ActRIIa protein exhibits dual activity, having bothcatabolic and anabolic effects. Thus, the disclosure establishes thatantagonists of the activin-ActRIIa signaling pathway may be used toincrease bone density and promote bone growth. While soluble ActRIIa mayaffect bone through a mechanism other than activin antagonism, thedisclosure nonetheless demonstrates that desirable therapeutic agentsmay be selected on the basis of an activin-ActRIIa antagonist activity.Therefore, in certain embodiments, the disclosure provides methods forusing activin-ActRIIa antagonists, including, for example,activin-binding ActRIIa polypeptides, anti-activin antibodies,anti-ActRIIa antibodies, activin- or ActRIIa-targeted small moleculesand aptamers, and nucleic acids that decrease expression of activin andActRIIa, to treat disorders associated with low bone density or low bonestrength, such as osteoporosis, or to promote bone growth in patients inneed thereof, such as in patients having a bone fracture. Additionally,the soluble ActRIIa polypeptide promotes bone growth without causing aconsistently measurable increase in muscle mass

In certain aspects, the disclosure provides polypeptides comprising asoluble, activin-binding ActRIIa polypeptide that binds to activin.ActRIIa polypeptides may be formulated as a pharmaceutical preparationcomprising the activin-binding ActRIIa polypeptide and apharmaceutically acceptable carrier. Preferably, the activin-bindingActRIIa polypeptide binds to activin with a K_(D) less than 1 micromolaror less than 100, 10 or 1 nanomolar. Optionally, the activin-bindingActRIIa polypeptide selectively binds activin versus GDF11 and/or GDF8,and preferably with a K_(D) that is at least 10-fold, 20-fold or 50-foldlower with respect to activin than with respect to GDF11 and/or GDF8.While not wishing to be bound to a particular mechanism of action, it isexpected that this degree of selectivity for activin inhibition overGDF11/GDF8 inhibition accounts for the selective effect on bone withouta consistently measurable effect on muscle. In many embodiments, anActRIIa polypeptide will be selected for causing less than 15%, lessthan 10% or less than 5% increase in muscle at doses that achievedesirable effects on bone. Preferably the composition is at least 95%pure, with respect to other polypeptide components, as assessed by sizeexclusion chromatography, and more preferably, the composition is atleast 98% pure. An activin-binding ActRIIa polypeptide for use in such apreparation may be any of those disclosed herein, such as a polypeptidehaving an amino acid sequence selected from SEQ ID NOs: 2, 3, 7 or 12,or having an amino acid sequence that is at least 80%, 85%, 90%, 95%,97% or 99% identical to an amino acid sequence selected from SEQ ID NOs:2, 3, 7, 12 or 13. An activin-binding ActRIIa polypeptide may include afunctional fragment of a natural ActRIIa polypeptide, such as onecomprising at least 10, 20 or 30 amino acids of a sequence selected fromSEQ ID NOs: 1-3 or a sequence of SEQ ID NO: 2, lacking the C-terminal 10to 15 amino acids (the “tail”).

A soluble, activin-binding ActRIIa polypeptide may include one or morealterations in the amino acid sequence (e.g., in the ligand-bindingdomain) relative to a naturally occurring ActRIIa polypeptide. Examplesof altered ActRIIa polypeptides are provided in WO 2006/012627, pp.59-60, incorporated by reference herein. The alteration in the aminoacid sequence may, for example, alter glycosylation of the polypeptidewhen produced in a mammalian, insect or other eukaryotic cell or alterproteolytic cleavage of the polypeptide relative to the naturallyoccurring ActRIIa polypeptide.

An activin-binding ActRIIa polypeptide may be a fusion protein that has,as one domain, an ActRIIa polypeptide (e.g., a ligand-binding portion ofan ActRIIa) and one or more additional domains that provide a desirableproperty, such as improved pharmacokinetics, easier purification,targeting to particular tissues, etc. For example, a domain of a fusionprotein may enhance one or more of in vivo stability, in vivo half life,uptake/administration, tissue localization or distribution, formation ofprotein complexes, multimerization of the fusion protein, and/orpurification. An activin-binding ActRIIa fusion protein may include animmunoglobulin Fc domain (wild-type or mutant) or a serum albumin orother polypeptide portion that provides desirable properties such asimproved pharmacokinetics, improved solubility or improved stability. Ina preferred embodiment, an ActRIIa-Fc fusion comprises a relativelyunstructured linker positioned between the Fc domain and theextracellular ActRIIa domain. This unstructured linker may correspond tothe roughly 15 amino acid unstructured region at the C-terminal end ofthe extracellular domain of ActRIIa (the “tail”), or it may be anartificial sequence of 1, 2, 3, 4 or 5 amino acids or a length ofbetween 5 and 15, 20, 30, 50 or more amino acids that are relativelyfree of secondary structure, or a mixture of both. A linker may be richin glycine and proline residues and may, for example, contain a singlesequence of threonine/serine and glycines or repeating sequences ofthreonine/serine and glycines (e.g., TG₄ or SG₄ singlets or repeats). Afusion protein may include a purification subsequence, such as anepitope tag, a FLAG tag, a polyhistidine sequence, and a GST fusion.Optionally, a soluble ActRIIa polypeptide includes one or more modifiedamino acid residues selected from: a glycosylated amino acid, aPEGylated amino acid, a farnesylated amino acid, an acetylated aminoacid, a biotinylated amino acid, an amino acid conjugated to a lipidmoiety, and an amino acid conjugated to an organic derivatizing agent. Apharmaceutical preparation may also include one or more additionalcompounds such as a compound that is used to treat a bone disorder.Preferably, a pharmaceutical preparation is substantially pyrogen free.In general, it is preferable that an ActRIIa protein be expressed in amammalian cell line that mediates suitably natural glycosylation of theActRIIa protein so as to diminish the likelihood of an unfavorableimmune response in a patient. Human and CHO cell lines have been usedsuccessfully, and it is expected that other common mammalian expressionsystems will be useful.

As described herein, ActRIIa proteins designated ActRIIa-Fc (a form witha minimal linker between the ActRIIa portion and the Fc portion) havedesirable properties, including selective binding to activin versus GDF8and/or GDF11, high affinity ligand binding and serum half life greaterthan two weeks in animal models. In certain embodiments the inventionprovides ActRIIa-Fc polypeptides and pharmaceutical preparationscomprising such polypeptides and a pharmaceutically acceptableexcipient.

In certain aspects, the disclosure provides nucleic acids encoding asoluble activin-binding ActRIIa polypeptide. An isolated polynucleotidemay comprise a coding sequence for a soluble, activin-binding ActRIIapolypeptide, such as described above. For example, an isolated nucleicacid may include a sequence coding for an extracellular domain (e.g.,ligand-binding domain) of an ActRIIa and a sequence that would code forpart or all of the transmembrane domain and/or the cytoplasmic domain ofan ActRIIa, but for a stop codon positioned within the transmembranedomain or the cytoplasmic domain, or positioned between theextracellular domain and the transmembrane domain or cytoplasmic domain.For example, an isolated polynucleotide may comprise a full-lengthActRIIa polynucleotide sequence such as SEQ ID NO: 4 or 5, or apartially truncated version, said isolated polynucleotide furthercomprising a transcription termination codon at least six hundrednucleotides before the 3′-terminus or otherwise positioned such thattranslation of the polynucleotide gives rise to an extracellular domainoptionally fused to a truncated portion of a full-length ActRIIa. Apreferred nucleic acid sequence is SEQ ID NO:14. Nucleic acids disclosedherein may be operably linked to a promoter for expression, and thedisclosure provides cells transformed with such recombinantpolynucleotides. Preferably the cell is a mammalian cell such as a CHOcell.

In certain aspects, the disclosure provides methods for making asoluble, activin-binding ActRIIa polypeptide. Such a method may includeexpressing any of the nucleic acids (e.g., SEQ ID NO: 4, 5 or 14)disclosed herein in a suitable cell, such as a Chinese hamster ovary(CHO) cell. Such a method may comprise: a) culturing a cell underconditions suitable for expression of the soluble ActRIIa polypeptide,wherein said cell is transformed with a soluble ActRIIa expressionconstruct; and b) recovering the soluble ActRIIa polypeptide soexpressed. Soluble ActRIIa polypeptides may be recovered as crude,partially purified or highly purified fractions. Purification may beachieved by a series of purification steps, including, for example, one,two or three or more of the following, in any order: protein Achromatography, anion exchange chromatography (e.g., Q sepharose),hydrophobic interaction chromatography (e.g., phenylsepharose), sizeexclusion chromatography, and cation exchange chromatography.

In certain aspects, an activin-ActRIIa antagonist disclosed herein, suchas a soluble, activin-binding ActRIIa polypeptide, may be used in amethod for promoting bone growth or increasing bone density in asubject. In certain embodiments, the disclosure provides methods fortreating a disorder associated with low bone density, or to promote bonegrowth, in patients in need thereof. A method may comprise administeringto a subject in need thereof an effective amount of activin-ActRIIaantagonist. In certain aspects, the disclosure provides uses ofactivin-ActRIIa antagonist for making a medicament for the treatment ofa disorder or condition as described herein.

In certain aspects, the disclosure provides a method for identifying anagent that stimulates growth of, or increased mineralization of, bone.The method comprises: a) identifying a test agent that binds to activinor a ligand-binding domain of an ActRIIa polypeptide; and b) evaluatingthe effect of the agent on growth of, or mineralization of, bone.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows the purification of ActRIIa-hFc expressed in CHO cells. Theprotein purifies as a single, well-defined peak.

FIG. 2 shows the binding of ActRIIa-hFc to activin and GDF-11, asmeasured by BiaCore™ assay.

FIG. 3 shows a schematic for the A-204 Reporter Gene Assay. The figureshows the Reporter vector: pGL3(CAGA)12 (described in Dennler et al,1998, EMBO 17: 3091-3100.) The CAGA12 motif is present in TGF-Betaresponsive genes (PAI-1 gene), so this vector is of general use forfactors signaling through Smad 2 and 3.

FIG. 4 shows the effects of ActRIIa-hFc (diamonds) and ActRIIa-mFc(squares) on GDF-8 signaling in the A-204 Reporter Gene Assay. Bothproteins exhibited substantial inhibition of GDF-8 mediated signaling atpicomolar concentrations.

FIG. 5 shows the effects of three different preparations of ActRIIa-hFcon GDF-11 signaling in the A-204 Reporter Gene Assay.

FIG. 6 shows examples of DEXA images of control- and ActRIIa-mFc-treatedBALB/c mice, before (top panels) and after (bottom panels) the 12-weektreatment period. Paler shading indicates increased bone density.

FIG. 7 shows a quantification of the effects of ActRIIa-mFc on honemineral density in BALB/c mice over the 12-week period. Treatments werecontrol (diamonds), 2 mg/kg dosing of ActRIIa-mFc (squares), 6 mg/kgdosing of ActRIIa-mFc (triangles) and 10 mg/kg dosing of ActRIIa-mFc(circles).

FIG. 8 shows a quantification of the effects of ActRIIa-mFc on bonemineral content in BALB/c mice over the 12-week period. Treatments werecontrol (diamonds), 2 mg/kg dosing of ActRIIa-mFc (squares), 6 mg/kgdosing of ActRIIa-mFc (triangles) and 10 mg/kg dosing of ActRIIa-mFc(circles).

FIG. 9 shows a quantification of the effects of ActRIIa-mFc on bonemineral density of the trabecular bone in ovariectomized (OVX) or shamoperated (SHAM) C57BL6 mice over after a 6-week period. Treatments werecontrol (PBS) or 10 mg/kg dosing of ActRIIa-mFc (ActRIIa).

FIG. 10 shows a quantification of the effects of ActRIIa-mFc on thetrabecular bone in ovariectomized (OVX) C57BL6 mice over a 12-weekperiod. Treatments were control (PBS; pale bars) or 10 mg/kg dosing ofActRIIa-mFc (ActRIIa, dark bars).

FIG. 11 shows a quantification of the effects of ActRIIa-mFc on thetrabecular bone in sham operated C57BL6 mice after 6 or 12 weeks oftreatment period. Treatments were control (PBS; pale bars) or 10 mg/kgdosing of ActRIIa-mFc (ActRIIa; dark bars).

FIG. 12 shows the results of pQCT analysis of bone density inovariectomized mice over 12 weeks of treatment. Treatments were control(PBS; pale bars) or ActRIIa-mFc (dark bars). y-axis: mg/ccm

FIG. 13 depicts the results of pQCT analysis of bone density in shamoperated mice over 12 weeks of treatment. Treatments were control (PBS;pale bars) or ActRIIa-mFc (dark bars), y-axis; mg/ccm

FIGS. 14A and 14B show whole body DEXA analysis after 12 weeks oftreatment (A) and ex vivo analysis of femurs (B). Light areas depictareas of high bone density.

FIG. 15 shows ex vivo pQCT analysis of the femoral midshaft after twelveweeks of treatment. Treatments were vehicle control (PBS, dark bars) andActRIIa-mFc (pale bars). The four bars to the left show total bonedensity while the four bars to the right show cortical bone density. Thefirst pair of bars in each set of four bars represent data fromovariectomized mice while the second pair of bars represent data fromsham operated mice.

FIG. 16 shows ex vivo pQCT analysis and diaphyseal bone content of thefemoral midshaft after twelve weeks of treatment. Treatments werevehicle control (PBS, dark bars) or ActRIIa-mFc (pale bars). The fourbars to the left show total bone content while the four bars to theright show cortical bone content. The first pair of bars in each set offour bars represent data from ovariectomized mice while the second pairof bars represent data from sham operated mice.

FIG. 17 shows ex vivo pQCT analysis of the femoral midshaft and femoralcortical thickness. Treatments were control (PBS, dark bars) andActRIIa-mFc (pale bars). The four bars to the left show endostealcircumference while the four bars to the right show periostealcircumference. The first pair of bars in each set of four bars representdata from ovariectomized mice while the second pair of bars representdata from sham operated mice.

FIG. 18 depicts the results of mechanical testing of femurs after twelveweeks of treatment. Treatments were control (PBS, dark bars) andActRIIa-mFc (pale bars). The two bars to the left represent data fromovariectomized mice while the last two bars represent data from shamoperated mice.

FIG. 19 shows the effects of ActRIIa-mFc on trabecular bone volume.

FIG. 20 shows the effects of ActRIIa-mFc on trabecular architecture inthe distal femur.

FIG. 21 shows the effects of ActRIIa-mFc on cortical bone.

FIG. 22 shows the effects of ActRIIa-mFc on the mechanical strength ofbone.

FIG. 23 shows the effects of different doses of ActRIIa-mFc on bonecharacteristics at three different dosages.

FIG. 24 shows bone histomorphometry indicating that ActRIIa-mFc has dualanabolic and anti-resorptive activity.

DETAILED DESCRIPTION OF THE INVENTION

1. Overview

The transforming growth factor-beta (TGF-beta) superfamily contains avariety of growth factors that share common sequence elements andstructural motifs. These proteins are known to exert biological effectson a large variety of cell types in both vertebrates and invertebrates.Members of the superfamily perform important functions during embryonicdevelopment in pattern formation and tissue specification and caninfluence a variety of differentiation processes, includingadipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis,neurogenesis, and epithelial cell differentiation. The family is dividedinto two general branches: the BMP/GDF and the TGF-beta/Activin/BMP10branches, whose members have diverse, often complementary effects. Bymanipulating the activity of a member of the TGF-beta family, it isoften possible to cause significant physiological changes in anorganism. For example, the Piedmontese and Belgian Blue cattle breedscarry a loss-of-function mutation in the GDF8 (also called myostatin)gene that causes a marked increase in muscle mass. Grobet et al., NatGenet. 1997, 17(1):71-4. Furthermore, in humans, inactive alleles ofGDF8 are associated with increased muscle mass and, reportedly,exceptional strength. Schuelke et al., N Engl J Med 2004, 350:2682-8.

Activins are dimeric polypeptide growth factors that belong to theTGF-beta superfamily. There are three principle activin forms (A, B, andAB) that are homo/heterodimers of two closely related β subunits(β_(A)β_(A), β_(B)β_(B), and β_(A)β_(B)). The human genome also encodesan activin C and an activin E, which are primarily expressed in theliver. In the TGF-beta superfamily, activins are unique andmultifunctional factors that can stimulate hormone production in ovarianand placental cells, support neuronal cell survival, influencecell-cycle progress positively or negatively depending on cell type, andinduce mesodermal differentiation at least in amphibian embryos (DePaoloet al., 1991, Proc Soc Ep Biol Med. 198:500-512; Dyson et al., 1997,Curr Biol. 7:81-84; Woodruff, 1998, Biochem Pharmacol. 55:953-963).Moreover, erythroid differentiation factor (EDF) isolated from thestimulated human monocytic leukemic cells was found to be identical toactivin A (Murata et al., 1988, PNAS, 85:2434). It has been suggestedthat activin A acts as a natural, positive regulator of erythropoiesisin the bone marrow. In several tissues, activin signaling is antagonizedby its related heterodimer, inhibin. For example, during the release offollicle-stimulating hormone (FSH) from the pituitary, activin promotesFSH secretion and synthesis, while inhibin prevents FSH secretion andsynthesis. Other proteins that may regulate activin bioactivity and/orbind to activin include follistatin (FS), follistatin-related protein(FSRP), α₂-macroglobulin, Cerberus, and endoglin.

TGF-β signals are mediated by heteromeric complexes of type I and typeII serine/threonine kinase receptors, which phosphorylate and activatedownstream Smad proteins upon ligand stimulation (Massagué, 2000, Nat.Rev. Mol. Cell Biol. 1:169-178). These type I and type II receptors aretransmembrane proteins, composed of a ligand-binding extracellulardomain with cysteine-rich region, a transmembrane domain, and acytoplasmic domain with predicted serine/threonine specificity. Type Ireceptors are essential for signaling; and type II receptors arerequired for binding ligands and for expression of type I receptors.Type I and II activin receptors form a stable complex after ligandbinding, resulting in phosphorylation of type I receptors by type IIreceptors.

Two related type II receptors, ActRIIa and ActRIIb, have been identifiedas the type II receptors for activins (Mathews and Vale, 1991, Cell65:973-982.; Attisano et al., 1992, Cell 68: 97-108). Besides activins,ActRIIa and ActRIIb can biochemically interact with several other TGF-βfamily proteins, including BMP7, Nodal, GDF8, and GDF11 (Yamashita etal., 1995, J. Cell Biol. 130:217-226; Lee and McPherron, 2001, Proc.Natl. Acad. Sci. 98:9306-9311; Yeo and Whitman, 2001, Mol. Cell 7:949-957; Oh et al., 2002, Genes Dev. 16:2749-54). ALK4 is the primarytype I receptor for activins, particularly for activin A, and ALK-7 mayserve as a receptor for activins as well, particularly for activin B.

As demonstrated herein, a soluble ActRIIa polypeptide (sActRIIa), whichshows substantial preference in binding to activin A as opposed to otherTGF-beta family members, such as GDF8 or GDF11, is effective to promotebone growth and increase bone density in vivo. While not wishing to bebound to any particular mechanism, it is expected that the effect ofsActRIIa is caused primarily by an activin antagonist effect, given thevery strong activin binding (picomolar dissociation constant) exhibitedby the particular sActRIIa construct used in these studies. Regardlessof mechanism, it is apparent from the data presented herein thatActRIIa-activin antagonists do increase bone density in normal mice andin mouse models for osteoporosis. It should be noted that bone is adynamic tissue, with growth or shrinkage and increased or decreaseddensity depending on a balance of factors that produce bone andstimulate mineralization (primarily osteoblasts) and factors thatdestroy and demineralize bone (primarily osteoclasts). Bone growth andmineralization may be increased by increasing the productive factors, bydecreasing the destructive factors, or both. The terms “promote bonegrowth” and “increase bone mineralization” refer to the observablephysical changes in bone and are intended to be neutral as to themechanism by which changes in bone occur.

The mouse models for osteoporosis and bone growth/density that were usedin the studies described herein are considered to be highly predictiveof efficacy in humans, and therefore, this disclosure provides methodsfor using ActRIIa polypeptides and other activin-ActRIIa antagonists topromote bone growth and increase bone density in humans. Activin-ActRIIaantagonists include, for example, activin-binding soluble ActRIIapolypeptides, antibodies that bind to activin (particularly the activinA or B subunits, also referred to as βA or βB) and disrupt ActRIIabinding, antibodies that bind to ActRIIa and disrupt activin binding,non-antibody proteins selected for activin or ActRIIa binding (see e.g.,WO/2002/088171, WO/2006/055689, WO/2002/032925, WO/2005/037989, US2003/0133939, and US 2005/0238646 for examples of such proteins andmethods for design and selection of same), randomized peptides selectedfor activin or ActRIIa binding, often affixed to an Fc domain. Twodifferent proteins (or other moieties) with activin or ActRIIa bindingactivity, especially activin binders that block the type I (e.g., asoluble type I activin receptor) and type II (e.g., a soluble type IIactivin receptor) binding sites, respectively, may be linked together tocreate a bifunctional binding molecule. Nucleic acid aptamers, smallmolecules and other agents that inhibit the activin-ActRIIa signalingaxis. Various proteins have activin-ActRIIa antagonist activity,including inhibin (i.e., inhibin alpha subunit), although inhibin doesnot universally antagonize activin in all tissues, follistatin (e.g.,follistatin-288 and follistatin-315), Cerberus, FSRP, endoglin, activinC, alpha(2)-macroglobulin, and an M108A (methionine to alanine change atposition 108) mutant activin A. Generally, alternative forms of activin,particularly those with alterations in the type I receptor bindingdomain can bind to type II receptors and fail to form an active ternarycomplex, thus acting as antagonists. Additionally, nucleic acids, suchas antisense molecules, siRNAs or ribozymes that inhibit activin A, B, Cor E, or, particularly, ActRIIa expression, can be used asactivin-ActRIIa antagonists. Preferably, the activin-ActRIIa antagonistto be used will exhibit selectivity for inhibiting activin-mediatedsignaling versus other members of the TGF-beta family, and particularlywith respect to GDF8 and GDF11. Soluble ActRIIb proteins do bind toactivin, however, the wild type protein does not exhibit significantselectivity in binding to activin versus GDF8/11, and preliminaryexperiments suggest that this protein does not provide the desiredeffects on bone, while also causing substantial muscle growth. However,altered forms of ActRIIb with different binding properties have beenidentified (see, e.g., WO 2006/012627, pp. 55-59, incorporated herein byreference) and these proteins may achieve the desired effects on bone.Native or altered ActRIIb may be given added specificity for activin bycoupling with a second, activin-selective binding agent.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them. The scope or meaning of any useof a term will be apparent from the specific context in which the termis used.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Typically, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values.

Alternatively, and particularly in biological systems, the terms “about”and “approximately” may mean values that are within an order ofmagnitude, preferably within 5-fold and more preferably within 2-fold ofa given value. Numerical quantities given herein are approximate unlessstated otherwise, meaning that the term “about” or “approximately” canbe inferred when not expressly stated.

The methods of the invention may include steps of comparing sequences toeach other, including wild-type sequence to one or more mutants(sequence variants). Such comparisons typically comprise alignments ofpolymer sequences, e.g., using sequence alignment programs and/oralgorithms that are well known in the art (for example, BLAST, FASTA andMEGALIGN, to name a few). The skilled artisan can readily appreciatethat, in such alignments, where a mutation contains a residue insertionor deletion, the sequence alignment will introduce a “gap” (typicallyrepresented by a dash, or “A”) in the polymer sequence not containingthe inserted or deleted residue.

“Homologous,” in all its grammatical forms and spelling variations,refers to the relationship between two proteins that possess a “commonevolutionary origin,” including proteins from superfamilies in the samespecies of organism, as well as homologous proteins from differentspecies of organism. Such proteins (and their encoding nucleic acids)have sequence homology, as reflected by their sequence similarity,whether in terms of percent identity or by the presence of specificresidues or motifs and conserved positions.

The term “sequence similarity,” in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin.

However, in common usage and in the instant application, the term“homologous,” when modified with an adverb such as “highly,” may referto sequence similarity and may or may not relate to a commonevolutionary origin.

2. ActRIIa Polypeptides

In certain aspects, the present invention relates to ActRIIapolypeptides. As used herein, the term “ActRIIa” refers to a family ofactivin receptor type IIa (ActRIIa) proteins from any species andvariants derived from such ActRIIa proteins by mutagenesis or othermodification. Reference to ActRIIa herein is understood to be areference to any one of the currently identified forms. Members of theActRIIa family are generally transmembrane proteins, composed of aligand-binding extracellular domain with a cysteine-rich region, atransmembrane domain, and a cytoplasmic domain with predictedserine/threonine kinase activity.

The term “ActRIIa polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of an ActRIIa family member as well asany variants thereof (including mutants, fragments, fusions, andpeptidomimetic forms) that retain a useful activity. For example,ActRIIa polypeptides include polypeptides derived from the sequence ofany known ActRIIa having a sequence at least about 80% identical to thesequence of an ActRIIa polypeptide, and preferably at least 85%, 90%,95%, 97%, 99% or greater identity. For example, an ActRIIa polypeptideof the invention may bind to and inhibit the function of an ActRIIaprotein and/or activin. Preferably, an ActRIIa polypeptide promotes bonegrowth and bone mineralization. Examples of ActRIIa polypeptides includehuman ActRIIa precursor polypeptide (SEQ ID NO: 1) and soluble humanActRIIa polypeptides (e.g., SEQ D NOs: 2, 3, 7 and 12).

The human ActRIIa precursor protein sequence is as follows:

(SEQ ID NO: 1) MGAAAKLAFAVFLISCSSGA ILGRSETQECLFFNANWEKDRT N QTGVEPCYGDKDKRRHCFATWK N ISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPYYNILLYSLVPLMLIAGIVICAFWVYRHHKMAYPPVLVPTQDPGPPPPSPLLGLKPLQLLEVKARGRFGCVWKAQLLNEYVAVKIFPIQDKQSWQNEYEVYSLPGMKHENILQFIGAEKRGTSVDVDLWLITAFHEKGSLSDFLKANVVSWNELCHIAETMARGLAYLHEDIPGLKDGHKPAISHRDIKSKNVLLKNNLTACIADFGLALKFEAGKSAGDTHGQVGTRRYMAPEVLEGAINFQRDAFLRIDMYAMGLVLWELASRCTAADGPVDEYMLPFEEEIGQHPSLEDMQEVVVHKKKRPVLRDYWQKHAGMAMLCETIEECWDHDAEARLSAGCVGERITQMQRLTNIITTEDIVTVVTMVTNVDFPPKESSL

The signal peptide is single underlined; the extracellular domain boldand the potential N-linked glycosylation sites are double underlined.

The human ActRIIa soluble (extracellular), processed polypeptidesequence is as follows:

(SEQ ID NO: 2) ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM EVTQPTSNPVTPKPP

The C-terminal “tail” of the extracellular domain is underlined. Thesequence with the “tail” deleted (a Δ15 sequence) is as follows:

(SEQ ID NO: 3) ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEM

The nucleic acid sequence encoding human ActRIIa precursor protein is asfollows (nucleotides 164-1705 of Genbank entry NM_001616):

(SEQ ID NO: 4) ATGGGAGCTGCTGCAAAGTTGGCGTTTGCCGTCTTTCTTATCTCCTGTTCTTCAGGTGCTATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTTCTTTAATGCTAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGGTGACAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGTTCCATTGAAATAGTGAAACAAGGTTGTTGGCTGGATGATATCAACTGCTATGACAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTTTGTTGCTGTGAGGGCAATATGTGTAATGAAAAGTTTTCTTATTTTCCAGAGATGGAAGTCACACAGCCCACTTCAAATCCAGTTACACCTAAGCCACCCTATTACAACATCCTGCTCTATTCCTTGGTGCCACTTATGTTAATTGCGGGGATTGTCATTTGTGCATTTTGGGTGTACAGGCATCACAAGATGGCCTACCCTCCTGTACTTGTTCCAACTCAAGACCCAGGACCACCCCCACCTTCTCCATTACTAGGGTTGAAACCACTGCAGTTATTAGAAGTGAAAGCAAGGGGAAGATTTGGTTGTGTCTGGAAAGCCCAGTTGCTTAACGAATATGTGGCTGTCAAAATATTTCCAATACAGGACAAACAGTCATGGCAAAATGAATACGAAGTCTACAGTTTGCCTGGAATGAAGCATGAGAACATATTACAGTTCATTGGTGCAGAAAAACGAGGCACCAGTGTTGATGTGGATCTTTGGCTGATCACAGCATTTCATGAAAAGGGTTCACTATCAGACTTTCTTAAGGCTAATGTGGTCTCTTGGAATGAACTGTGTCATATTGCAGAAACCATGGCTAGAGGATTGGCATATTTACATGAGGATATACCTGGCCTAAAAGATGGCCACAAACCTGCCATATCTCACAGGGACATCAAAAGTAAAAATGTGCTGTTGAAAAACAACCTGACAGCTTGCATTGCTGACTTTGGGTTGGCCTTAAAATTTGAGGCTGGCAAGTCTGCAGGCGATACCCATGGACAGGTTGGTACCCGGAGGTACATGGCTCCAGAGGTATTAGAGGGTGCTATAAACTTCCAAAGGGATGCATTTTTGAGGATAGATATGTATGCCATGGGATTAGTCCTATGGGAACTGGCTTCTCGCTGTACTGCTGCAGATGGACCTGTAGATGAATACATGTTGCCATTTGAGGAGGAAATTGGCCAGCATCCATCTCTTGAAGACATGCAGGAAGTTGTTGTGCATAAAAAAAAGAGGCCTGTTTTAAGAGATTATTGGCAGAAACATGCTGGAATGGCAATGCTCTGTGAAACCATTGAAGAATGTTGGGATCACGACGCAGAAGCCAGGTTATCAGCTGGATGTGTAGGTGAAAGAATTACCCAGATGCAGAGACTAACAAATATTATTACCACAGAGGACATTGTAACAGTGGTCACAATGGTGACAAATGTTGACTTTCCTCCCAAAGAATCTAGTCTATGA

The nucleic acid sequence encoding a human ActRIIa soluble(extracellular is as follows:

(SEQ ID NO: 5) ATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTTCTTTAATGCTAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGGTGACAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGTTCCATTGAAATAGTGAAACAAGGTTGTTGGCTGGATGATATCAACTGCTATGACAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTTTGTTGCTGTGAGGGCAATATGTGTAATGAAAAGTTTTCTTATTTTCCAGAGATGGAAGTCACACAGCCCACTTCAAATCCAGTTACACCTAAGCCACCC

In a specific embodiment, the invention relates to soluble ActRIIapolypeptides. As described herein, the term “soluble ActRIIapolypeptide” generally refers to polypeptides comprising anextracellular domain of an ActRIIa protein. The term “soluble ActRIIapolypeptide,” as used herein, includes any naturally occurringextracellular domain of an ActRIIa protein as well as any variantsthereof (including mutants, fragments and peptidomimetic forms. Anactivin-binding ActRIIa polypeptide is one that retains the ability tobind to activin, particularly activin AA, AB or BB. Preferably, anactivin-binding ActRIIa polypeptide will bind to activin AA with adissociation constant of 1 nM or less. Amino acid sequences of humanActRIIa precursor protein is provided below. The extracellular domain ofan ActRIIa protein binds to activin and is generally soluble, and thuscan be termed a soluble, activin-binding ActRIIa polypeptide. Examplesof soluble, activin-binding ActRIIa polypeptides include the solublepolypeptide illustrated in SEQ ID NOs: 2, 3, 7, 12 and 13. SEQ ID NO:7is referred to as ActRIIa-hFc, and is described further in the Examples.Other examples of soluble, activin-binding ActRIIa polypeptides comprisea signal sequence in addition to the extracellular domain of an ActRIIaprotein, for example, the honey bee mellitin leader sequence (SEQ ID NO:8), the tissue plaminogen activator (TPA) leader (SEQ ID NO: 9) or thenative ActRIIa leader (SEQ ID NO: 10). The ActRIIa-hFc polypeptideillustrated in SEQ NO:13 uses a TPA leader.

Functionally active fragments of ActRIIa polypeptides can be obtained byscreening polypeptides recombinantly produced from the correspondingfragment of the nucleic acid encoding an ActRIIa polypeptide. Inaddition, fragments can be chemically synthesized using techniques knownin the art such as conventional Merrifield solid phase f-Moc or t-Bocchemistry. The fragments can be produced (recombinantly or by chemicalsynthesis) and tested to identify those peptidyl fragments that canfunction as antagonists (inhibitors) of ActRIIa protein or signalingmediated by activin.

Functionally active variants of ActRIIa polypeptides can be obtained byscreening libraries of modified polypeptides recombinantly produced fromthe corresponding mutagenized nucleic acids encoding an ActRIIapolypeptide. The variants can be produced and tested to identify thosethat can function as antagonists (inhibitors) of ActRIIa protein orsignaling mediated by activin. In certain embodiments, a functionalvariant of the ActRIIa polypeptides comprises an amino acid sequencethat is at least 75% identical to an amino acid sequence selected fromSEQ ID NOs: 2 or 3. In certain cases, the functional variant has anamino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%identical to an amino acid sequence selected from SEQ ID NOs: 2 or 3.

Functional variants may be generated by modifying the structure of anActRIIa polypeptide for such purposes as enhancing therapeutic efficacy,or stability (e.g., ex vivo shelf life and resistance to proteolyticdegradation in vivo). Such modified ActRIIa polypeptides when selectedto retain activin binding, are considered functional equivalents of thenaturally-occurring ActRIIa polypeptides. Modified ActRIIa polypeptidescan also be produced, for instance, by amino acid substitution,deletion, or addition. For instance, it is reasonable to expect that anisolated replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, a threonine with a serine, or a similarreplacement of an amino acid with a structurally related amino acid(e.g., conservative mutations) will not have a major effect on thebiological activity of the resulting molecule. Conservative replacementsare those that take place within a family of amino acids that arerelated in their side chains. Whether a change in the amino acidsequence of an ActRIIa polypeptide results in a functional homolog, canbe readily determined by assessing the ability of the variant ActRIIapolypeptide to produce a response in cells in a fashion similar to thewild-type ActRIIa polypeptide.

In certain embodiments, the present invention contemplates specificmutations of the ActRIIa polypeptides so as to alter the glycosylationof the polypeptide. Such mutations may be selected so as to introduce oreliminate one or more glycosylation sites, such as O-linked or N-linkedglycosylation sites. Asparagine-linked glycosylation recognition sitesgenerally comprise a tripeptide sequence, asparagine-X-threonine (orasparagines-X-serine) (where “X” is any amino acid) which isspecifically recognized by appropriate cellular glycosylation enzymes.The alteration may also be made by the addition of, or substitution by,one or more serine or threonine residues to the sequence of thewild-type ActRIIa polypeptide (for O-linked glycosylation sites). Avariety of amino acid substitutions or deletions at one or both of thefirst or third amino acid positions of a glycosylation recognition site(and/or amino acid deletion at the second position) results innon-glycosylation at the modified tripeptide sequence. Another means ofincreasing the number of carbohydrate moieties on an ActRIIa polypeptideis by chemical or enzymatic coupling of glycosides to the ActRIIapolypeptide. Depending on the coupling mode used, the sugar(s) may beattached to (a) arginine and histidine; (b) free carboxyl groups; (c)free sulfhydryl groups such as those of cysteine; (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline; (e)aromatic residues such as those of phenylalanine, tyrosine, ortryptophan; or (f) the amide group of glutamine. These methods aredescribed in WO 87/05330 published Sep. 11, 1987, and in Aplin andWriston (1981) CRC Crit. Rev. Biochem., pp. 259-306, incorporated byreference herein. Removal of one or more carbohydrate moieties presenton an ActRIIa polypeptide may be accomplished chemically and/orenzymatically. Chemical deglycosylation may involve, for example,exposure of the ActRIIa polypeptide to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving the aminoacid sequence intact. Chemical deglycosylation is further described byHakimuddin et al. (1987) Arch. Biochem. Biophys. 259:52 and by Edge etal. (1981) Anal. Biochem. 118:131, Enzymatic cleavage of carbohydratemoieties on ActRIIa polypeptides can be achieved by the use of a varietyof endo- and exo-glycosidases as described by Thotakura et al. (1987)Meth. Enzymol. 138:350. The sequence of an ActRIIa polypeptide may beadjusted, as appropriate, depending on the type of expression systemused, as mammalian, yeast, insect and plant cells may all introducediffering glycosylation patterns that can be affected by the amino acidsequence of the peptide. In general, ActRIIa proteins for use in humanswill be expressed in a mammalian cell line that provides properglycosylation, such as HEK293 or CHO cell lines, although othermammalian expression cell lines, yeast cell lines with engineeredglycosylation enzymes and insect cells are expected to be useful aswell.

This disclosure further contemplates a method of generating mutants,particularly sets of combinatorial mutants of an ActRIIa polypeptide, aswell as truncation mutants; pools of combinatorial mutants areespecially useful for identifying functional variant sequences. Thepurpose of screening such combinatorial libraries may be to generate,for example, ActRIIa polypeptide variants which can act as eitheragonists or antagonist, or alternatively, which possess novel activitiesall together. A variety of screening assays are provided below, and suchassays may be used to evaluate variants. For example, an ActRIIapolypeptide variant may be screened for ability to bind to an ActRIIaligand, to prevent binding of an ActRIIa ligand to an ActRIIapolypeptide or to interfere with signaling caused by an ActRIIa ligand.

The activity of an ActRIIa polypeptide or its variants may also betested in a cell-based or in vivo assay. For example, the effect of anActRIIa polypeptide variant on the expression of genes involved in boneproduction or bone destruction may be assessed. This may, as needed, beperformed in the presence of one or more recombinant ActRIIa ligandproteins (e.g., activin), and cells may be transfected so as to producean ActRIIa polypeptide and/or variants thereof, and optionally, anActRIIa ligand. Likewise, an ActRIIa polypeptide may be administered toa mouse or other animal, and one or more bone properties, such asdensity or volume may be assessed. The healing rate for bone fracturesmay also be evaluated. Dual-energy x-ray absorptiometry (DEXA) is awell-established, non-invasive, quantitative technique for assessingbone density in an animal. In humans central DEXA systems may be used toevaluate bone density in the spine and pelvis. These are the bestpredictors of overall bone density. Peripheral DEXA systems may be usedto evaluate bone density in peripheral bones, including, for example,the bones of the hand, wrist, ankle and foot. Traditional x-ray imagingsystems, including CAT scans, may be used to evaluate bone growth andfracture healing. The mechanical strength of bone may also be evaluated.

Combinatorially-derived variants can be generated which have a selectiveor generally increased potency relative to a naturally occurring ActRIIapolypeptide. Likewise, mutagenesis can give rise to variants which haveintracellular half-lives dramatically different than the corresponding awild-type ActRIIa polypeptide. For example, the altered protein can berendered either more stable or less stable to proteolytic degradation orother cellular processes which result in destruction of, or otherwiseinactivation of a native ActRIIa polypeptide. Such variants, and thegenes which encode them, can be utilized to alter ActRIIa polypeptidelevels by modulating the half-life of the ActRIIa polypeptides. Forinstance, a short half-life can give rise to more transient biologicaleffects and can allow tighter control of recombinant ActRIIa polypeptidelevels within the patient. In an Fc fusion protein, mutations may bemade in the linker (if any) and/or the Fc portion to alter the half-lifeof the protein.

A combinatorial library may be produced by way of a degenerate libraryof genes encoding a library of polypeptides which each include at leasta portion of potential ActRIIa polypeptide sequences. For instance, amixture of synthetic oligonucleotides can be enzymatically ligated intogene sequences such that the degenerate set of potential ActRIIapolypeptide nucleotide sequences are expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(e.g., for phage display).

There are many ways by which the library of potential homologs can begenerated from a degenerate oligonucleotide sequence. Chemical synthesisof a degenerate gene sequence can be carried out in an automatic DNAsynthesizer, and the synthetic genes then be ligated into an appropriatevector for expression. The synthesis of degenerate oligonucleotides iswell known in the art (see for example, Narang, S A (1983) Tetrahedron39:3; Itakura et al., (1981) Recombinant DNA, Proc. 3rd ClevelandSympos. Macromolecules, ed. A G Walton, Amsterdam: Elsevier pp 273-289;Itakura et al., (1984) Annu. Rev. Biochem. 53:323; Itakura et al.,(1984) Science 198:1056; Ike et al., (1983) Nucleic Acid Res. 11:477).Such techniques have been employed in the directed evolution of otherproteins (see, for example, Scott et al., (1990) Science 249:386-390;Roberts et al., (1992) PNAS USA 89:2429-2433; Devlin et al., (1990)Science 249: 404-406; Cwirla et (1990) PNAS USA 87: 6378-6382; as wellas U.S. Pat. Nos. 5,223,409, 5,198,346, and 5,096,815).

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library. For example, ActRIIa polypeptide variants can begenerated and isolated from a library by screening using, for example,alanine scanning mutagenesis and the like (Ruf et al., (1994)Biochemistry 33:1565-1572; Wang et al., (1994) J. Biol. Chem.269:3095-3099; Balint et al., (1993) Gene 137:109-118; Grodberg et al.,(1993) Eur. J. Biochem. 218:597-601; Nagashima et al., (1993) J. Biol.Chem. 268:2888-2892; Lowman et al., (1991) Biochemistry 30:10832-10838;and Cunningham et al., (1989) Science 244:1081-1085), by linker scanningmutagenesis (Gostin et al., (1993) Virology 193:653-660; Brown et al.,(1992) Mol. Cell Biol. 12:2644-2652; McKnight et al., (1982) Science232:316); by saturation mutagenesis (Meyers et al., (1986) Science232:613); by PCR mutagenesis (Leung et al., (1989) Method Cell Mol Biol1:11-19); or by random mutagenesis, including chemical mutagenesis, etc.(Miller et al., (1992.) A Short Course in Bacterial Genetics, CSHLPress, Cold Spring Harbor, N.Y.; and Greener et al., (1994) Strategiesin Mol Biol 7:32-34). Linker scanning mutagenesis, particularly in acombinatorial setting, is an attractive method for identifying truncated(bioactive) forms of ActRIIa polypeptides.

A wide range of techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations andtruncations, and, for that matter, for screening cDNA libraries for geneproducts having a certain property. Such techniques will be generallyadaptable for rapid screening of the gene libraries generated by thecombinatorial mutagenesis of ActRIIa polypeptides. The most widely usedtechniques for screening large gene libraries typically comprisescloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected. Preferredassays include activin binding assays and activin-mediated cellsignaling assays.

In certain embodiments, the ActRIIa polypeptides of the invention mayfurther comprise post-translational modifications in addition to anythat are naturally present in the ActRIIa polypeptides. Suchmodifications include, but are not limited to, acetylation,carboxylation, glycosylation, phosphorylation, lipidation, andacylation. As a result, the modified ActRIIa polypeptides may containnon-amino acid elements, such as polyethylene glycols, lipids, poly- ormono-saccharide, and phosphates. Effects of such non-amino acid elementson the functionality of a ActRIIa polypeptide may be tested as describedherein for other ActRIIa polypeptide variants. When an ActRIIapolypeptide is produced in cells by cleaving a nascent form of theActRIIa polypeptide, post-translational processing may also be importantfor correct folding and/or function of the protein. Different cells(such as CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293) have specificcellular machinery and characteristic mechanisms for suchpost-translational activities and may be chosen to ensure the correctmodification and processing of the ActRIIa polypeptides.

In certain aspects, functional variants or modified forms of the ActRIIapolypeptides include fusion proteins having at least a portion of theActRIIa polypeptides and one or more fusion domains. Well known examplesof such fusion domains include, but are not limited to, polyhistidine,Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A,protein G, an immunoglobulin heavy chain constant region (Fc), maltosebinding protein (MBP), or human serum albumin. A fusion domain may beselected so as to confer a desired property. For example, some fusiondomains are particularly useful for isolation of the fusion proteins byaffinity chromatography. For the purpose of affinity purification,relevant matrices for affinity chromatography, such as glutathione-,amylase-, and nickel- or cobalt-conjugated resins are used. Many of suchmatrices are available in “kit” form, such as the Pharmacia GSTpurification system and the QIAexpress™ system (Qiagen) useful with(HIS₆) fusion partners. As another example, a fusion domain may beselected so as to facilitate detection of the ActRIIa polypeptides.Examples of such detection domains include the various fluorescentproteins (e.g., GFP) as well as “epitope tags,” which are usually shortpeptide sequences for which a specific antibody is available. Well knownepitope tags for which specific monoclonal antibodies are readilyavailable include FLAG, influenza virus haemagglutinin (HA), and c-myctags. In some cases, the fusion domains have a protease cleavage site,such as for Factor Xa or Thrombin, which allows the relevant protease topartially digest the fusion proteins and thereby liberate therecombinant proteins therefrom. The liberated proteins can then beisolated from the fusion domain by subsequent chromatographicseparation. In certain preferred embodiments, an ActRIIa polypeptide isfused with a domain that stabilizes the ActRIIa polypeptide in vivo (a“stabilizer” domain). By “stabilizing” is meant anything that increasesserum half life, regardless of whether this is because of decreaseddestruction, decreased clearance by the kidney, or other pharmacokineticeffect. Fusions with the Fc portion of an immunoglobulin are known toconfer desirable pharmacokinetic properties on a wide range of proteins.Likewise, fusions to human serum albumin can confer desirableproperties. Other types of fusion domains that may be selected includemultimerizing (e.g., dimerizing, tetramerizing) domains and functionaldomains (that confer an additional biological function, such as furtherstimulation of bone growth or muscle growth, as desired).

As a specific example, the present invention provides a fusion proteincomprising a soluble extracellular domain of ActRIIa fused to an Fcdomain (e.g., SEQ ID NO: 6).

THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD(A)VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK(A)VSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN(A)HYTQKSLSLPGK*

Optionally, the Fc domain has one or more mutations at residues such asAsp-265, lysine 322, and Asn-434. In certain cases, the mutant Fc domainhaving one or more of these mutations (e.g., Asp-265 mutation) hasreduced ability of binding to the Fcγ receptor relative to a wildtype Fcdomain. In other cases, the mutant Fc domain having one or more of thesemutations (e.g., Asn-434 mutation) has increased ability of binding tothe MHC class I-related Fc-receptor (FcRN) relative to a wildtype Fcdomain.

It is understood that different elements of the fusion proteins may bearranged in any manner that is consistent with the desiredfunctionality. For example, an ActRIIa polypeptide may be placedC-terminal to a heterologous domain, or, alternatively, a heterologousdomain may be placed C-terminal to an ActRIIa polypeptide. The ActRIIapolypeptide domain and the heterologous domain need not be adjacent in afusion protein, and additional domains or amino acid sequences may beincluded C- or N-terminal to either domain or between the domains.

In certain embodiments, the ActRIIa polypeptides of the presentinvention contain one or more modifications that are capable ofstabilizing the ActRIIa polypeptides. For example, such modificationsenhance the in vitro half life of the ActRIIa polypeptides, enhancecirculatory half life of the ActRIIa polypeptides or reduce proteolyticdegradation of the ActRIIa polypeptides. Such stabilizing modificationsinclude, but are not limited to, fusion proteins (including, forexample, fusion proteins comprising an ActRIIa polypeptide and astabilizer domain), modifications of a glycosylation site (including,for example, addition of a glycosylation site to an ActRIIapolypeptide), and modifications of carbohydrate moiety (including, forexample, removal of carbohydrate moieties from an ActRIIa polypeptide).In the case of fusion proteins, an ActRIIa polypeptide is fused to astabilizer domain such as an IgG molecule (e.g., an Fc domain). As usedherein, the term “stabilizer domain” not only refers to a fusion domain(e.g., Fc) as in the case of fusion proteins, but also includesnonproteinaceous modifications such as a carbohydrate moiety, ornonproteinaceous polymer, such as polyethylene glycol.

In certain embodiments, the present invention makes available isolatedand/or purified forms of the ActRIIa polypeptides, which are isolatedfrom, or otherwise substantially free of, other proteins. ActRIIapolypeptides will generally be produced by expression from recombinantnucleic acids.

3. Nucleic Acids Encoding ActRIIa Polypeptides

In certain aspects, the invention provides isolated and/or recombinantnucleic acids encoding any of the ActRIIa polypeptides (e.g., solubleActRIIa polypeptides), including fragments, functional variants andfusion proteins disclosed herein. For example, SEQ ID NO: 4 encodes thenaturally occurring human ActRIIa precursor polypeptide, while SEQ IDNO: 5 encodes the processed extracellular domain of ActRIIa. The subjectnucleic acids may be single stranded or double stranded. Such nucleicacids may be DNA or RNA molecules. These nucleic acids may be used, forexample, in methods for making ActRIIa polypeptides or as directtherapeutic agents (e.g., in a gene therapy approach).

In certain aspects, the subject nucleic acids encoding ActRIIapolypeptides are further understood to include nucleic acids that arevariants of SEQ ID NO: 4 or 5. Variant nucleotide sequences includesequences that differ by one or more nucleotide substitutions, additionsor deletions, such as allelic variants.

In certain embodiments, the invention provides isolated or recombinantnucleic acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%,99% or 100% identical to SEQ ID NO: 4 or 5. One of ordinary skill in theart will appreciate that nucleic acid sequences complementary to SEQ IDNO: 4 or 5, and variants of SEQ ID NO: 4 or 5 are also within the scopeof this invention. In further embodiments, the nucleic acid sequences ofthe invention can be isolated, recombinant, and/or fused with aheterologous nucleotide sequence, or in a DNA library.

In other embodiments, nucleic acids of the invention also includenucleotide sequences that hybridize under highly stringent conditions tothe nucleotide sequence designated in SEQ ID NO: 4 or 5, complementsequence of SEQ ID NO: 4 or 5, or fragments thereof. As discussed above,one of ordinary skill in the art will understand readily thatappropriate stringency conditions which promote DNA hybridization can bevaried. One of ordinary skill in the art will understand readily thatappropriate stringency conditions which promote DNA hybridization can bevaried. For example, one could perform the hybridization at 6.0× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by a wash of2.0×SSC at 50° C. For example, the salt concentration in the wash stepcan be selected from a low stringency of about 2.0×SSC at 50° C. to ahigh stringency of about 0.2×SSC at 50° C. In addition, the temperaturein the wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or temperature or saltconcentration may be held constant while the other variable is changed.In one embodiment, the invention provides nucleic acids which hybridizeunder low stringency conditions of 6×SSC at room temperature followed bya wash at 2×SSC at room temperature.

Isolated nucleic acids which differ from the nucleic acids as set forthin SEQ NOs: 4 or 5 due to degeneracy in the genetic code are also withinthe scope of the invention. For example, a number of amino acids aredesignated by more than one triplet. Codons that specify the same aminoacid, or synonyms (for example, CAU and CAC are synonyms for histidine)may result in “silent” mutations which do not affect the amino acidsequence of the protein. However, it is expected that DNA sequencepolymorphisms that do lead to changes in the amino acid sequences of thesubject proteins will exist among mammalian cells. One skilled in theart will appreciate that these variations in one or more nucleotides (upto about 3-5% of the nucleotides) of the nucleic acids encoding aparticular protein may exist among individuals of a given species due tonatural allelic variation. Any and all such nucleotide variations andresulting amino acid polymorphisms are within the scope of thisinvention.

In certain embodiments, the recombinant nucleic acids of the inventionmay be operably linked to one or more regulatory nucleotide sequences inan expression construct. Regulatory nucleotide sequences will generallybe appropriate to the host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the invention. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome. In a preferred embodiment, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selectable marker genes are well known in the art and willvary with the host cell used.

In certain aspects of the invention, the subject nucleic acid isprovided in an expression vector comprising a nucleotide sequenceencoding an ActRIIa polypeptide and operably linked to at least oneregulatory sequence. Regulatory sequences are art-recognized and areselected to direct expression of the ActRIIa polypeptide. Accordingly,the term regulatory sequence includes promoters, enhancers, and otherexpression control elements. Exemplary regulatory sequences aredescribed in Goeddel; Gene Expression Technology: Methods in Enzymology,Academic Press, San Diego, Calif. (1990). For instance, any of a widevariety of expression control sequences that control the expression of aDNA sequence when operatively linked to it may be used in these vectorsto express DNA sequences encoding an ActRIIa polypeptide. Such usefulexpression control sequences, include, for example, the early and latepromoters of SV40, tet promoter, adenovirus or cytomegalovirus immediateearly promoter, RSV promoters, the lac system, the trp system, the TACor TRC system, T7 promoter whose expression is directed by T7 RNApolymerase, the major operator and promoter regions of phage lambda thecontrol regions for fd coat protein, the promoter for 3-phosphoglyceratekinase or other glycolytic enzymes, the promoters of acid phosphatase,e.g., Pho5, the promoters of the yeast α-mating factors, the polyhedronpromoter of the baculovirus system and other sequences known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof. It should be understood thatthe design of the expression vector may depend on such factors as thechoice of the host cell to be transformed and/or the type of proteindesired to be expressed. Moreover, the vector's copy number, the abilityto control that copy number and the expression of any other proteinencoded by the vector, such as antibiotic markers, should also beconsidered.

A recombinant nucleic acid of the invention can be produced by ligatingthe cloned gene, or a portion thereof, into a vector suitable forexpression in either prokaryotic cells, eukaryotic cells (yeast, avian,insect or mammalian), or both. Expression vehicles for production of arecombinant ActRIIa polypeptide include plasmids and other vectors. Forinstance, suitable vectors include plasmids of the types: pBR322-derivedplasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derivedplasmids and pUC-derived plasmids for expression in prokaryotic cells,such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription units that are expressed in eukaryotic cells.The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found below inthe description of gene therapy delivery systems. The various methodsemployed in the preparation of the plasmids and in transformation ofhost organisms are well known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures, see Molecular Cloning A Laboratory Manual, 3rdEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press, 2001). In some instances, it may be desirable toexpress the recombinant polypeptides by the use of a baculovirusexpression system. Examples of such baculovirus expression systemsinclude pVL-derived vectors (such as pVL1392, pVL1393 and pVL941),pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors(such as the ß-gal containing pBlueBac III).

In a preferred embodiment, a vector will be designed for production ofthe subject ActRIIa polypeptides in CHO cells, such as a Pcmv-Scriptvector (Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen,Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison, Wis.). As willbe apparent, the subject gene constructs can be used to cause expressionof the subject ActRIIa polypeptides in cells propagated in culture,e.g., to produce proteins, including fusion proteins or variantproteins, for purification.

This disclosure also pertains to a host cell transfected with arecombinant gene including a coding sequence (e.g., SEQ ID NO: 4 or 5)for one or more of the subject ActRIIa polypeptides. The host cell maybe any prokaryotic or eukaryotic cell. For example, an ActRIIapolypeptide of the invention may be expressed in bacterial cells such asE. coli, insect cells (e.g., using a baculovirus expression system),yeast, or mammalian cells. Other suitable host cells are known to thoseskilled in the art.

Accordingly, the present invention further pertains to methods ofproducing the subject ActRIIa polypeptides. For example, a host celltransfected with an expression vector encoding an ActRIIa polypeptidecan be cultured under appropriate conditions to allow expression of theActRIIa polypeptide to occur. The ActRIIa polypeptide may be secretedand isolated from a mixture of cells and medium containing the ActRIIapolypeptide. Alternatively, the ActRIIa polypeptide may be retainedcytoplasmically or in a membrane fraction and the cells harvested, lysedand the protein isolated. A cell culture includes host cells, media andother byproducts. Suitable media for cell culture are well known in theart. The subject ActRIIa polypeptides can be isolated from cell culturemedium, host cells, or both, using techniques known in the art forpurifying proteins, including ion-exchange chromatography, gelfiltration chromatography, ultrafiltration, electrophoresis,immunoaffinity purification with antibodies specific for particularepitopes of the ActRIIa polypeptides and affinity purification with anagent that binds to a domain fused to the ActRIIa polypeptide (e.g., aprotein A column may be used to purify an ActRIIa-Fc fusion). In apreferred embodiment, the ActRIIa polypeptide is a fusion proteincontaining a domain which facilitates its purification. In a preferredembodiment, purification is achieved by a series of columnchromatography steps, including, for example, three or more of thefollowing, in any order: protein A chromatography, Q sepharosechromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange. Asdemonstrated herein, ActRIIa-hFc protein was purified to a purityof >98% as determined by size exclusion chromatography and >95% asdetermined by SDS PAGE. This level of purity was sufficient to achievedesirable effects on bone in mice and an acceptable safety profile inmice, rats and non-human primates.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant ActRIIapolypeptide, can allow purification of the expressed fusion protein byaffinity chromatography using a Ni²⁺ metal resin. The purificationleader sequence can then be subsequently removed by treatment withenterokinase to provide the purified ActRIIa polypeptide (e.g., seeHochuli et al., (1987) J. Chromatography 411:177; and Janknecht et al.,PNAS USA 88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons: 1992).

4. Alternative Activin and ActRIIa Antagonists

The data presented herein demonstrates that antagonists ofactivin-ActRIIa signaling can be used to promote bone growth and bonemineralization. Although soluble ActRIIa polypeptides, and particularlyActRIIa-Fc, are preferred antagonists, and although such antagonists mayaffect bone through a mechanism other than activin antagonism (e.g.,activin inhibition may be an indicator of the tendency of an agent toinhibit the activities of a spectrum of molecules, including, perhaps,other members of the TGF-beta superfamily, and such collectiveinhibition may lead to the desired effect on bone), other types ofactivin-ActRIIa antagonists are expected to be useful, includinganti-activin (e.g., A, B, C or E) antibodies, anti-ActRIIa antibodies,antisense, RNAi or ribozyme nucleic acids that inhibit the production ofActRIIa and other inhibitors of activin or ActRIIa, particularly thosethat disrupt activin-ActRIIa binding.

An antibody that is specifically reactive with an ActRIIa polypeptide(e.g., a soluble ActRIIa polypeptide) and which either bindscompetitively to ligand with the ActRIIa polypeptide or otherwiseinhibits ActRIIa-mediated signaling may be used as an antagonist ofActRIIa polypeptide activities. Likewise, an antibody that isspecifically reactive with an activin A polypeptide and which disruptsActRIIa binding may be used as an antagonist.

By using immunogens derived from an ActRIIa polypeptide or an activinpolypeptide, anti-protein/anti-peptide antisera or monoclonal antibodiescan be made by standard protocols (see, for example, Antibodies: ALaboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press:1988)). A mammal, such as a mouse, a hamster or rabbit can be immunizedwith an immunogenic form of the ActRIIa polypeptide, an antigenicfragment which is capable of eliciting an antibody response, or a fusionprotein. Techniques for conferring immunogenicity on a protein orpeptide include conjugation to carriers or other techniques well knownin the art. An immunogenic portion of an ActRIIa or activin polypeptidecan be administered in the presence of adjuvant. The progress ofimmunization can be monitored by detection of antibody titers in plasmaor serum. Standard ELISA or other immunoassays can be used with theimmunogen as antigen to assess the levels of antibodies.

Following immunization of an animal with an antigenic preparation of anActRIIa polypeptide, antisera can be obtained and, if desired,polyclonal antibodies can be isolated from the serum. To producemonoclonal antibodies, antibody-producing cells (lymphocytes) can beharvested from an immunized animal and fused by standard somatic cellfusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, andinclude, for example, the hybridoma technique (originally developed byKohler and Milstein, (1975) Nature, 256: 495-497), the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss.Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with an ActRIIapolypeptide and monoclonal antibodies isolated from a culture comprisingsuch hybridoma cells.

The term “antibody” as used herein is intended to include fragmentsthereof which are also specifically reactive with a subject polypeptide.Antibodies can be fragmented using conventional techniques and thefragments screened for utility in the same manner as described above forwhole antibodies. For example, F(ab)₂ fragments can be generated bytreating antibody with pepsin. The resulting F(ab)₂ fragment can betreated to reduce disulfide bridges to produce Fab fragments. Theantibody of the present invention is further intended to includebispecific, single-chain, chimeric, humanized and fully human moleculeshaving affinity for an ActRIIa or activin polypeptide conferred by atleast one CDR region of the antibody. An antibody may further comprise alabel attached thereto and able to be detected (e.g., the label can be aradioisotope, fluorescent compound, enzyme or enzyme co-factor).

In certain embodiments, the antibody is a recombinant antibody, whichterm encompasses any antibody generated in part by techniques ofmolecular biology, including CDR-grafted or chimeric antibodies, humanor other antibodies assembled from library-selected antibody domains,single chain antibodies and single domain antibodies (e.g., human V_(H)proteins or camelid V_(HH) proteins). In certain embodiments, anantibody of the invention is a monoclonal antibody, and in certainembodiments, the invention makes available methods for generating novelantibodies. For example, a method for generating a monoclonal antibodythat binds specifically to an ActRIIa polypeptide or activin polypeptidemay comprise administering to a mouse an amount of an immunogeniccomposition comprising the antigen polypeptide effective to stimulate adetectable immune response, obtaining antibody-producing cells (e.g.,cells from the spleen) from the mouse and fusing the antibody-producingcells with myeloma cells to obtain antibody-producing hybridomas, andtesting the antibody-producing hybridomas to identify a hybridoma thatproduces a monocolonal antibody that binds specifically to the antigen.Once obtained, a hybridoma can be propagated in a cell culture,optionally in culture conditions where the hybridoma-derived cellsproduce the monoclonal antibody that binds specifically to the antigen.The monoclonal antibody may be purified from the cell culture.

The adjective “specifically reactive with” as used in reference to anantibody is intended to mean, as is generally understood in the art,that the antibody is sufficiently selective between the antigen ofinterest (e.g., an ActRIIa polypeptide) and other antigens that are notof interest that the antibody is useful for, at minimum, detecting thepresence of the antigen of interest in a particular type of biologicalsample. In certain methods employing the antibody, such as therapeuticapplications, a higher degree of specificity in binding may bedesirable. Monoclonal antibodies generally have a greater tendency (ascompared to polyclonal antibodies) to discriminate effectively betweenthe desired antigens and cross-reacting polypeptides. One characteristicthat influences the specificity of an antibody:antigen interaction isthe affinity of the antibody for the antigen. Although the desiredspecificity may be reached with a range of different affinities,generally preferred antibodies will have an affinity (a dissociationconstant) of about 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ or less. Given theextraordinarily tight binding between activin and ActRIIa, it isexpected that a neutralizing anti-activin or anti-ActRIIa antibody wouldgenerally have a dissociation constant of 10⁻¹⁰ or less.

In addition, the techniques used to screen antibodies in order toidentify a desirable antibody may influence the properties of theantibody obtained. For example, if an antibody is to be used for bindingan antigen in solution, it may be desirable to test solution binding. Avariety of different techniques are available for testing interactionbetween antibodies and antigens to identify particularly desirableantibodies. Such techniques include ELISAs, surface plasmon resonancebinding assays (e.g., the Biacore™ binding assay, Biacore AB, Uppsala,Sweden), sandwich assays (e.g., the paramagnetic bead system of IGENInternational, Inc., Gaithersburg, Md.), western blots,immunoprecipitation assays, and immunohistochemistry.

Examples of categories of nucleic acid compounds that are activin orActRIIa antagonists include anti sense nucleic acids, RNAi constructsand catalytic nucleic acid constructs. A nucleic acid compound may besingle or double stranded. A double stranded compound may also includeregions of overhang or non-complementarity, where one or the other ofthe strands is single stranded. A single stranded compound may includeregions of self-complementarity, meaning that the compound forms aso-called “hairpin” or “stem-loop” structure, with a region of doublehelical structure. A nucleic acid compound may comprise a nucleotidesequence that is complementary to a region consisting of no more than1000, no more than 500, no more than 250, no more than 100 or no morethan 50, 35, 30, 25, 22, 20 or 18 nucleotides of the full-length ActRIIanucleic acid sequence or activin βA or activin βB nucleic acid sequence.The region of complementarity will preferably be at least 8 nucleotides,and optionally at least 10 or at least 15 nucleotides, and optionallybetween 15 and 25 nucleotides. A region of complementarity may fallwithin an intron, a coding sequence or a noncoding sequence of thetarget transcript, such as the coding sequence portion. Generally, anucleic acid compound will have a length of about 8 to about 500nucleotides or base pairs in length, and optionally the length will beabout 14 to about 50 nucleotides. A nucleic acid may be a DNA(particularly for use as an antisense), RNA or RNA:DNA hybrid. Any onestrand may include a mixture of DNA and RNA, as well as modified formsthat cannot readily be classified as either DNA or RNA. Likewise, adouble stranded compound may be DNA:DNA, DNA:RNA or RNA:RNA, and any onestrand may also include a mixture of DNA and RNA, as well as modifiedforms that cannot readily be classified as either DNA or RNA. A nucleicacid compound may include any of a variety of modifications, includingone or modifications to the backbone (the sugar-phosphate portion in anatural nucleic acid, including internucleotide linkages) or the baseportion (the purine or pyrimidine portion of a natural nucleic acid). Anantisense nucleic acid compound will preferably have a length of about15 to about 30 nucleotides and will often contain one or moremodifications to improve characteristics such as stability in the serum,in a cell or in a place where the compound is likely to be delivered,such as the stomach in the case of orally delivered compounds and thelung for inhaled compounds. In the case of an RNAi construct, the strandcomplementary to the target transcript will generally be RNA ormodifications thereof. The other strand may be RNA, DNA or any othervariation. The duplex portion of double stranded or single stranded“hairpin” RNAi construct will preferably have a length of 18 to 40nucleotides in length and optionally about 21 to 23 nucleotides inlength, so long as it serves as a Dicer substrate. Catalytic orenzymatic nucleic acids may be ribozymes or DNA enzymes and may alsocontain modified forms. Nucleic acid compounds may inhibit expression ofthe target by about 50%, 75%, 90% or more when contacted with cellsunder physiological conditions and at a concentration where a nonsenseor sense control has little or no effect. Preferred concentrations fortesting the effect of nucleic acid compounds are 1, 5 and 10 micromolar.Nucleic acid compounds may also be tested for effects on, for example,bone growth and mineralization.

5. Screening Assays

In certain aspects, the present invention relates to the use of ActRIIapolypeptides soluble ActRIIa polypeptides) and activin polypeptides toidentify compounds (agents) which are agonist or antagonists of theactivin-ActRIIa signaling pathway. Compounds identified through thisscreening can be tested to assess their ability to modulate bone growthor mineralization in vitro. Optionally, these compounds can further betested in animal models to assess their ability to modulate tissuegrowth in vivo.

There are numerous approaches to screening for therapeutic agents formodulating tissue growth by targeting activin and ActRIIa polypeptides.In certain embodiments, high-throughput screening of compounds can becarried out to identify agents that perturb activin or ActRIIa-mediatedeffects on bone. In certain embodiments, the assay is carried out toscreen and identify compounds that specifically inhibit or reducebinding of an ActRIIa polypeptide to activin. Alternatively, the assaycan be used to identify compounds that enhance binding of an ActRIIapolypeptide to activin. In a further embodiment, the compounds can beidentified by their ability to interact with an activin or ActRIIapolypeptide.

A variety of assay formats will suffice and, in light of the presentdisclosure, those not expressly described herein will nevertheless becomprehended by one of ordinary skill in the art. As described herein,the test compounds (agents) of the invention may be created by anycombinatorial chemical method. Alternatively, the subject compounds maybe naturally occurring biomolecules synthesized in vivo or in vitro.Compounds (agents) to be tested for their ability to act as modulatorsof tissue growth can be produced, for example, by bacteria, yeast,plants or other organisms (e.g., natural products), produced chemically(e.g., small molecules, including peptidomimetics), or producedrecombinantly. Test compounds contemplated by the present inventioninclude non-peptidyl organic molecules, peptides, polypeptides,peptidomimetics, sugars, hormones, and nucleic acid molecules. In aspecific embodiment, the test agent is a small organic molecule having amolecular weight of less than about 2,000 daltons.

The test compounds of the invention can be provided as single, discreteentities, or provided in libraries of greater complexity, such as madeby combinatorial chemistry. These libraries can comprise, for example,alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers andother classes of organic compounds. Presentation of test compounds tothe test system can be in either an isolated form or as mixtures ofcompounds, especially in initial screening steps. Optionally, thecompounds may be optionally derivatized with other compounds and havederivatizing groups that facilitate isolation of the compounds.Non-limiting examples of derivatizing groups include biotin,fluorescein, digoxygenin, green fluorescent protein, isotopes,polyhistidine, magnetic beads, glutathione S transferase (GST),photoactivatible crosslinkers or any combinations thereof.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity or bioavailability of the test compound canbe generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity between an ActRIIapolypeptide and activin.

Merely to illustrate, in an exemplary screening assay of the presentinvention, the compound of interest is contacted with an isolated andpurified ActRIIa polypeptide which is ordinarily capable of binding toactivin. To the mixture of the compound and ActRIIa polypeptide is thenadded a composition containing an ActRIIa ligand. Detection andquantification of ActRIIa/activin complexes provides a means fordetermining the compound's efficacy at inhibiting (or potentiating)complex formation between the ActRIIa polypeptide and activin. Theefficacy of the compound can be assessed by generating dose responsecurves from data obtained using various concentrations of the testcompound. Moreover, a control assay can also be performed to provide abaseline for comparison. For example, in a control assay, isolated andpurified activin is added to a composition containing the ActRIIapolypeptide, and the formation of ActRIIa/activin complex is quantitatedin the absence of the test compound. It will be understood that, ingeneral, the order in which the reactants may be admixed can be varied,and can be admixed simultaneously. Moreover, in place of purifiedproteins, cellular extracts and lysates may be used to render a suitablecell-free assay system.

Complex formation between the ActRIIa polypeptide and activin may bedetected by a variety of techniques. For instance, modulation of theformation of complexes can be quantitated using, for example, detectablylabeled proteins such as radiolabeled (e.g., ³²P, ³⁵S, ¹⁴C or ³H),fluorescently labeled (e.g., FITC), or enzymatically labeled ActRIIapolypeptide or activin, by immunoassay, or by chromatographic detection.

In certain embodiments, the present invention contemplates the use offluorescence polarization assays and fluorescence resonance energytransfer (FRET) assays in measuring, either directly or indirectly, thedegree of interaction between an ActRIIa polypeptide and its bindingprotein. Further, other modes of detection, such as those based onoptical waveguides (PCT Publication WO 96/26432 and U.S. Pat. No.5,677,196), surface plasmon resonance (SPR), surface charge sensors, andsurface force sensors, are compatible with many embodiments of theinvention.

Moreover, the present invention contemplates the use of an interactiontrap assay, also known as the “two hybrid assay,” for identifying agentsthat disrupt or potentiate interaction between an ActRIIa polypeptideand its binding protein. See for example, U.S. Pat. No. 5,283,317;Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; andIwabuchi et al. (1993) Oncogene 8:1693-1696). In a specific embodiment,the present invention contemplates the use of reverse two hybrid systemsto identify compounds (e.g., small molecules or peptides) thatdissociate interactions between an ActRIIa polypeptide and its bindingprotein. See for example, Vidal and Legrain, (1999) Nucleic Acids Res27:919-29; Vidal and Legrain. (1999) Trends Biotechnol 17:374-81; andU.S. Pat. Nos. 5,525,490; 5,955,280; and 5,965,368.

In certain embodiments, the subject compounds are identified by theirability to interact with an ActRIIa or activin polypeptide of theinvention. The interaction between the compound and the ActRIIa oractivin polypeptide may be covalent or non-covalent. For example, suchinteraction can be identified at the protein level using in vitrobiochemical methods, including photo-crosslinking, radiolabeled ligandbinding, and affinity chromatography (Jakoby W B et al., 1974, Methodsin Enzymology 46: 1). In certain cases, the compounds may be screened ina mechanism based assay, such as an assay to detect compounds which bindto an activin or ActRIIa polypeptide. This may include a solid phase orfluid phase binding event. Alternatively, the gene encoding an activinor ActRIIa polypeptide can be transfected with a reporter system (e.g.,β-galactosidase, luciferase, or green fluorescent protein) into a celland screened against the library preferably by a high throughputscreening or with individual members of the library. Other mechanismbased binding assays may be used, for example, binding assays whichdetect changes in free energy. Binding assays can be performed with thetarget fixed to a well, bead or chip or captured by an immobilizedantibody or resolved by capillary electrophoresis. The bound compoundsmay be detected usually using colorimetric or fluorescence or surfaceplasmon resonance.

In certain aspects, the present invention provides methods and agentsfor modulating (stimulating or inhibiting) bone formation and increasingbone mass. Therefore, any compound identified can be tested in wholecells or tissues, in vitro or in vivo, to confirm their ability tomodulate bone growth or mineralization. Various methods known in the artcan be utilized for this purpose.

For example, the effect of the ActRIIa or activin polypeptides or testcompounds on bone or cartilage growth can be determined by measuringinduction of Msx2 or differentiation of osteoprogenitor cells intoosteoblasts in cell based assays (see, e.g., Daluiski et al., Nat Genet.2001, 27(1):84-8; Hino et al., Front Biosci. 2004, 9:1520-9). Anotherexample of cell-based assays includes analyzing the osteogenic activityof the subject ActRIIa or activin polypeptides and test compounds inmesenchymal progenitor and osteoblastic cells. To illustrate,recombinant adenoviruses expressing an activin or ActRIIa polypeptidecan be constructed to infect pluripotent mesenchymal progenitorC3H10T1/2 cells, preosteoblastic C2C12 cells, and osteoblastic TE-85cells. Osteogenic activity is then determined by measuring the inductionof alkaline phosphatase, osteocalcin, and matrix mineralization (see,e.g., Cheng et al., J bone Joint Surg Am. 2003, 85-A(8):1544-52).

The present invention also contemplates in vivo assays to measure boneor cartilage growth. For example, Namkung-Matthai et al., Bone, 28:80-86(2001) discloses a rat osteoporotic model in which bone repair duringthe early period after fracture is studied. Kubo et al., SteroidBiochemistry & Molecular Biology, 68:197-202 (1999) also discloses a ratosteoporotic model in which bone repair during the late period afterfracture is studied. Andersson et al., J. Endocrinol. 170:529-537describe a mouse osteoporosis model in which mice are ovariectomized,which causes the mice to lose substantial bone mineral content and bonemineral density, with the trabecular bone losing roughly 50% of bonemineral density. Bone density could be increased in the ovariectomizedmice by administration of factors such as parathyroid hormone. Incertain aspects, the present invention makes use of fracture healingassays that are known in the art. These assays include fracturetechnique, histological analysis, and biomechanical analysis, which aredescribed in, for example, U.S. Pat. No. 6,521,750, which isincorporated by reference in its entirety for its disclosure ofexperimental protocols for causing as well as measuring the extent offractures, and the repair process.

6. Exemplary Therapeutic Uses

In certain embodiments, activin-ActRIIa antagonists (e.g., ActRIIapolypeptides) of the present invention can be used for treating orpreventing a disease or condition that is associated with bone damage,whether, e.g., through breakage, loss or demineralization. In certainembodiments, the present invention provides methods of treating orpreventing bone damage in an individual in need thereof throughadministering to the individual a therapeutically effective amount of anactivin-ActRIIa antagonist, particularly an ActRIIa polypeptide. Incertain embodiments, the present invention provides methods of promotingbone growth or mineralization in an individual in need thereof throughadministering to the individual a therapeutically effective amount of anactivin-ActRIIa antagonist, particularly an ActRIIa polypeptide. Thesemethods are preferably aimed at therapeutic and prophylactic treatmentsof animals, and more preferably, humans. In certain embodiments, thedisclosure provides for the use of activin-ActRIIa antagonists(particularly soluble ActRIIa polypeptides and neutralizing antibodiestargeted to activin or ActRIIa) for the treatment of disordersassociated with low bone density or decreased bone strength.

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample. The term “treating” as used hereinincludes prophylaxis of the named condition or amelioration orelimination of the condition once it has been established. In eithercase, prevention or treatment may be discerned in the diagnosis providedby a physician and the intended result of administration of thetherapeutic agent.

The disclosure provides methods of inducing bone and/or cartilageformation, preventing bone loss, increasing bone mineralization orpreventing the demineralization of bone. For example, the subjectactivin-ActRIIa antagonists have application in treating osteoporosisand the healing of bone fractures and cartilage defects in humans andother animals. ActRIIa or activin polypeptides may be useful in patientsthat are diagnosed with subclinical low bone density, as a protectivemeasure against the development of osteoporosis.

In one specific embodiment, methods and compositions of the presentinvention may find medical utility in the healing of bone fractures andcartilage defects in humans and other animals. The subject methods andcompositions may also have prophylactic use in closed as well as openfracture reduction and also in the improved fixation of artificialjoints. De novo bone formation induced by an osteogenic agentcontributes to the repair of congenital, trauma-induced, or oncologicresection induced craniofacial defects, and also is useful in cosmeticplastic surgery. In certain cases, the subject activin-ActRIIaantagonists may provide an environment to attract bone-forming cells,stimulate growth of hone-forming cells or induce differentiation ofprogenitors of bone-forming cells. Activin-ActRIIa antagonists of theinvention may also be useful in the treatment of osteoporosis.

Methods and compositions of the invention can be applied to conditionscharacterized by or causing bone loss, such as osteoporosis (includingsecondary osteoporosis), hyperparathyroidism, Cushing's disease, Paget'sdisease, thyrotoxicosis, chronic diarrheal state or malabsorption, renaltubular acidosis, or anorexia nervosa.

Osteoporosis may be caused by, or associated with, various factors.Being female, particularly a post-menopausal female, having a low bodyweight, and leading a sedentary lifestyle are all risk factors forosteoporosis (loss of bone mineral density, leading to fracture risk).Persons having any of the following profiles may be candidates fortreatment with an ActRIIa antagonist: a post-menopausal woman and nottaking estrogen or other hormone replacement therapy; a person with apersonal or maternal history of hip fracture or smoking; apost-menopausal woman who is tall (over 5 feet 7 inches) or thin (lessthan 125 pounds); a man with clinical conditions associated with boneloss; a person using medications that are known to cause bone loss,including corticosteroids such as Prednisone™, various anti-seizuremedications such as Dilantin™ and certain barbiturates, or high-dosethyroid replacement drugs; a person having type 1 diabetes, liverdisease, kidney disease or a family history of osteoporosis; a personhaving high bone turnover (e.g., excessive collagen in urine samples); aperson with a thyroid condition, such as hyperthyroidism; a person whohas experienced a fracture after only mild trauma; a person who has hadx-ray evidence of vertebral fracture or other signs of osteoporosis.

As noted above, osteoporosis can also result as a condition associatedwith another disorder or from the use of certain medications.Osteoporosis resulting from drugs or another medical condition is knownas secondary osteoporosis. In a condition known as Cushing's disease,the excess amount of cortisol produced by the body results inosteoporosis and fractures. The most common medications associated withsecondary osteoporosis are the corticosteroids, a class of drugs thatact like cortisol, a hormone produced naturally by the adrenal glands.Although adequate levels of thyroid hormones (which are produced by thethyroid gland) are needed for the development of the skeleton, excessthyroid hormone can decrease bone mass over time. Antacids that containaluminum can lead to bone loss when taken in high doses by people withkidney problems, particularly those undergoing dialysis. Othermedications that can cause secondary osteoporosis include phenytoin(Dilantin) and barbiturates that are used to prevent seizures;methotrexate (Rheumatrex, Immunex, Folex PFS), a drug for some forms ofarthritis, cancer, and immune disorders; cyclosporine (Sandimmune,Neoral), a drug used to treat some autoimmune diseases and to suppressthe immune system in organ transplant patients; luteinizinghormone-releasing hormone agonists (Lupron, Zoladex), used to treatprostate cancer and endometriosis; heparin (Calciparine, Liquaemin), ananticlotting medication; and cholestyramine (Questran) and colestipol(Colestid), used to treat high cholesterol. Bone loss resulting fromcancer therapy is widely recognized and termed cancer therapy inducedbone loss (CTIBL). Bone metastases can create cavities in the bone thatmay be corrected by treatment with activin-ActRIIa antagonists.

In a preferred embodiment, activin-ActRIIa antagonists, particularly asoluble ActRIIa, disclosed herein may be used in cancer patients.Patients having certain tumors (e.g. prostate, breast, multiple myelomaor any tumor causing hyperparathyroidism) are at high risk for bone lossdue to tumor-induced bone loss as well as bone metastases andtherapeutic agents. Such patients may be treated with activin-ActRIIaantagonists even in the absence of evidence of bone loss or bonemetastases. Patients may also be monitored for evidence of bone loss orbone metastases, and may be treated with activin-ActRIIa antagonists inthe event that indicators suggest an increased risk. Generally, DEXAscans are employed to assess changes in bone density, while indicatorsof bone remodeling may be used to assess the likelihood of bonemetastases. Serum markers may be monitored. Bone specific alkalinephosphatase (BSAP) is an enzyme that is present in osteoblasts. Bloodlevels of BSAP are increased in patients with bone metastasis and otherconditions that result in increased bone remodeling. Osteocalcin andprocollagen peptides are also associated with bone formation and bonemetastases. Increases in BSAP have been detected in patients with bonemetastasis caused by prostate cancer, and to a lesser degree, in bonemetastases from breast cancer. Bone Morphogenetic Protein-7 (BMP-7)levels are high in prostate cancer that has metastasized to bone, butnot in bone metastases due to bladder, skin, liver, or lung cancer. Type1 Carboxy-terminal telopeptide (ICTP) is a crosslink found in collagenthat is formed during to the resorption of bone. Since bone isconstantly being broken down and reformed, ICTP will be found throughoutthe body. However, at the site of bone metastasis, the level will besignificantly higher than in an area of normal bone. ICTP has been foundin high levels in bone metastasis due to prostate, lung, and breastcancer. Another collagen crosslink, Type 1 N-terminal telopeptide (NTx),is produced along with ICTP during bone turnover. The amount of NTx isincreased in bone metastasis caused by many different types of cancerincluding lung, prostate, and breast cancer. Also, the levels of NTxincrease with the progression of the bone metastasis. Therefore, thismarker can be used to both detect metastasis as well as measure theextent of the disease. Other markers of resorption include pyridinolineand deoxypyridinoline. Any increase in resorption markers or markers ofbone metastases indicate the need for activin-ActRIIa antagonist therapyin a patient.

Activin-ActRIIa antagonists may be conjointly administered with otherpharmaceutical agents. Conjoint administration may be accomplished byadministration of a single co-formulation, by simultaneousadministration or by administration at separate times. Activin-ActRIIaantagonists may be particularly advantageous if administered with otherbone-active agents. A patient may benefit from conjointly receivingactivin-ActRIIa antagonist and taking calcium supplements, vitamin D,appropriate exercise and/or, in some cases, other medication. Examplesof other medications include, bisphosphonates (alendronate, ibandronateand risedronate), calcitonin, estrogens, parathyroid hormone andraloxifene. The bisphosphonates (alendronate, ibandronate andrisedronate), calcitonin, estrogens and raloxifene affect the boneremodeling cycle and are classified as anti-resorptive medications. Boneremodeling consists of two distinct stages: bone resorption and boneformation. Anti-resorptive medications slow or stop the bone-resorbingportion of the bone-remodeling cycle but do not slow the bone-formingportion of the cycle. As a result, new formation continues at a greaterrate than bone resorption, and bone density may increase over time.Teriparatide, a form of parathyroid hormone, increases the rate of boneformation in the bone remodeling cycle. Alendronate is approved for boththe prevention (5 mg per day or 35 mg once a week) and treatment (10 mgper day or 70 mg once a week) of postmenopausal osteoporosis.Alendronate reduces bone loss, increases bone density and reduces therisk of spine, wrist and hip fractures. Alendronate also is approved fortreatment of glucocorticoid-induced osteoporosis in men and women as aresult of long-term use of these medications (i.e., prednisone andcortisone) and for the treatment of osteoporosis in men. Alendronateplus vitamin D is approved for the treatment of osteoporosis inpostmenopausal women (70 mg once a week plus vitamin D), and fortreatment to improve bone mass in men with osteoporosis. Ibandronate isapproved for the prevention and treatment of postmenopausalosteoporosis. Taken as a once-a-month pill (150 mg), ibandronate shouldbe taken on the same day each month. Ibandronate reduces bone loss,increases bone density and reduces the risk of spine fractures.Risedronate is approved for the prevention and treatment ofpostmenopausal osteoporosis. Taken daily (5 mg dose) or weekly (35 mgdose or 35 mg dose with calcium), risedronate slows bone loss, increasesbone density and reduces the risk of spine and non-spine fractures.Risedronate also is approved for use by men and women to prevent and/ortreat glucocorticoid-induced osteoporosis that results from long-termuse of these medications (i.e., prednisone or cortisone). Calcitonin isa naturally occurring hormone involved in calcium regulation and bonemetabolism. In women who are more than 5 years beyond menopause,calcitonin slows bone loss, increases spinal bone density, and mayrelieve the pain associated with bone fractures. Calcitonin reduces therisk of spinal fractures. Calcitonin is available as an injection(50-100 IU daily) or nasal spray (200 IU daily). Estrogen therapy(ET)/Hormone therapy (HT) is approved for the prevention ofosteoporosis. ET has been shown to reduce bone loss, increase bonedensity in both the spine and hip, and reduce the risk of hip and spinalfractures in postmenopausal women. ET is administered most commonly inthe form of a pill or skin patch that delivers a low dose ofapproximately 0.3 mg daily or a standard dose of approximately 0.625 mgdaily and is effective even when started after age 70. When estrogen istaken alone, it can increase a woman's risk of developing cancer of theuterine lining (endometrial cancer). To eliminate this risk, healthcareproviders prescribe the hormone progestin in combination with estrogen(hormone replacement therapy or HT) for those women who have an intactuterus. ET/HT relieves menopause symptoms and has been shown to have abeneficial effect on bone health. Side effects may include vaginalbleeding, breast tenderness, mood disturbances and gallbladder disease.Raloxifene, 60 mg a day, is approved for the prevention and treatment ofpostmenopausal osteoporosis. It is from a class of drugs calledSelective Estrogen Receptor Modulators (SERMs) that have been developedto provide the beneficial effects of estrogens without their potentialdisadvantages. Raloxifene increases bone mass and reduces the risk ofspine fractures. Data are not yet available to demonstrate thatraloxifene can reduce the risk of hip and other non-spine fractures.Teriparatide, a form of parathyroid hormone, is approved for thetreatment of osteoporosis in postmenopausal women and men who are athigh risk for a fracture. This medication stimulates new bone formationand significantly increases bone mineral density. In postmenopausalwomen, fracture reduction was noted in the spine, hip, foot, ribs andwrist. In men, fracture reduction was noted in the spine, but there wereinsufficient data to evaluate fracture reduction at other sites.Teriparatide is self-administered as a daily injection for up to 24months.

7. Pharmaceutical Compositions

In certain embodiments, activin-ActRIIa antagonists (e.g., ActRIIapolypeptides) of the present invention are formulated with apharmaceutically acceptable carrier. For example, an ActRIIa polypeptidecan be administered alone or as a component of a pharmaceuticalformulation (therapeutic composition). The subject compounds may beformulated for administration in any convenient way for use in human orveterinary medicine.

In certain embodiments, the therapeutic method of the invention includesadministering the composition systemically, or locally as an implant ordevice. When administered, the therapeutic composition for use in thisinvention is, of course, in a pyrogen-free, physiologically acceptableform. Therapeutically useful agents other than the ActRIIa antagonistswhich may also optionally be included in the composition as describedabove, may be administered simultaneously or sequentially with thesubject compounds (e.g., ActRIIa polypeptides) in the methods of theinvention.

Typically, ActRIIa antagonists will be administered parentally.Pharmaceutical compositions suitable for parenteral administration maycomprise one or more ActRIIa polypeptides in combination with one ormore pharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

Further, the composition may be encapsulated or injected in a form fordelivery to a target tissue site (e.g., bone). In certain embodiments,compositions of the present invention may include a matrix capable ofdelivering one or more therapeutic compounds (e.g., ActRIIapolypeptides) to a target tissue site (e.g., bone), providing astructure for the developing tissue and optimally capable of beingresorbed into the body. For example, the matrix may provide slow releaseof the ActRIIa polypeptides. Such matrices may be formed of materialspresently in use for other implanted medical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the subjectcompositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid andpolyanhydrides. Other potential materials are biodegradable andbiologically well defined, such as bone or dermal collagen. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are non-biodegradable andchemically defined, such as sintered hydroxyapatite, bioglass,aluminates, or other ceramics. Matrices may be comprised of combinationsof any of the above mentioned types of material, such as polylactic acidand hydroxyapatite or collagen and tricalciumphosphate. The bioceramicsmay be altered in composition, such as in calcium-aluminate-phosphateand processing to alter pore size, particle size, particle shape, andbiodegradability.

In certain embodiments, methods of the invention can be administered fororally, e.g., in the form of capsules, cachets, pills, tablets, lozenges(using a flavored basis, usually sucrose and acacia or tragacanth),powders, granules, or as a solution or a suspension in an aqueous ornon-aqueous liquid, or as an oil-in-water or water-in-oil liquidemulsion, or as an elixir or syrup, or as pastilles (using an inertbase, such as gelatin and glycerin, or sucrose and acacia) and/or asmouth washes and the like, each containing a predetermined amount of anagent as an active ingredient. An agent may also be administered as abolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), one or more therapeuticcompounds of the present invention may be mixed with one or morepharmaceutically acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetylalcohol and glycerol monostearate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents such as ethoxylated isostearyl alcohols, polyoxyethylenesorbitol, and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

The compositions of the invention may also contain adjuvants, such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption, such as aluminum monostearate andgelatin.

It is understood that the dosage regimen will be determined by theattending physician considering various factors which modify the actionof the subject compounds of the invention (e.g., ActRIIa polypeptides).The various factors include, but are not limited to, amount of boneweight desired to be formed, the degree of bone density loss, the siteof bone damage, the condition of the damaged bone, the patient's age,sex, and diet, the severity of any disease that may be contributing tobone loss, time of administration, and other clinical factors.Optionally, the dosage may vary with the type of matrix used in thereconstitution and the types of compounds in the composition. Theaddition of other known growth factors to the final composition, mayalso affect the dosage. Progress can be monitored by periodic assessmentof bone growth and/or repair, for example, X-rays (including DEXA),histomorphometric determinations, and tetracycline labeling.

Experiments with mice have demonstrated that effects of ActRIIa-Fc onbone are detectable when the compound is dosed at intervals and amountssufficient to achieve serum concentrations of 0.2 μg/kg or greater, andserum levels of 1 μg/kg or 2 μg/kg or greater are desirable forachieving significant effects on bone density and strength. Althoughthere is no indication that higher doses of ActRIIa-Fc are undesirabledue to side effects, dosing regimens may be designed to reach serumconcentrations of between 0.2 and 15 μg/kg, and optionally between 1 and5 μg/kg. In humans, serum levels of 0.2 μg/kg may be achieved with asingle dose of 0.1 mg/kg or greater and serum levels of 1 μg/kg may beachieved with a single dose of 0.3 mg/kg or greater. The observed serumhalf-life of the molecule is between about 20 and 30 days, substantiallylonger than most Fc fusion proteins, and thus a sustained effectiveserum level may be achieved, for example, by dosing with 0.2-0.4 mg/kgon a weekly or biweekly basis, or higher doses may be used with longerintervals between dosings. For example, doses of 1-3 mg/kg might be usedon a monthly or bimonthly basis, and the effect on bone may besufficiently durable that dosing is necessary only once every 3, 4, 5,6, 9, 12 or more months.

In certain embodiments, the present invention also provides gene therapyfor the in vivo production of ActRIIa polypeptides. Such therapy wouldachieve its therapeutic effect by introduction of the ActRIIapolynucleotide sequences into cells or tissues having the disorders aslisted above. Delivery of ActRIIa polynucleotide sequences can beachieved using a recombinant expression vector such as a chimeric virusor a colloidal dispersion system. Preferred for therapeutic delivery ofActRIIa polynucleotide sequences is the use of targeted liposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. Retroviral vectors can be madetarget-specific by attaching, for example, a sugar, a glycolipid, or aprotein. Preferred targeting is accomplished by using an antibody. Thoseof skill in the art will recognize that specific polynucleotidesequences can be inserted into the retroviral genome or attached to aviral envelope to allow target specific delivery of the retroviralvector containing the ActRIIa polynucleotide. In a preferred embodiment,the vector is targeted to bone or cartilage.

Alternatively, tissue culture cells can be directly transfected withplasmids encoding the retroviral structural genes gag, pol and env, byconventional calcium phosphate transfection. These cells are thentransfected with the vector plasmid containing the genes of interest.The resulting cells release the retroviral vector into the culturemedium.

Another targeted delivery system for ActRIIa polynucleotides is acolloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. RNA, DNA andintact virions can be encapsulated within the aqueous interior and bedelivered to cells in a biologically active form (see e.g., Fraley, etal., Trends Biochem. Sci., 6:77, 1981). Methods for efficient genetransfer using a liposome vehicle, are known in the art, see e.g.,Mannino, et al., Biotechniques, 6:682, 1988. The composition of theliposome is usually a combination of phospholipids, usually incombination with steroids, especially cholesterol. Other phospholipidsor other lipids may also be used. The physical characteristics ofliposomes depend on pH, ionic strength, and the presence of divalentcations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Illustrative phospholipids include eggphosphatidylcholine, dipalmitoylphosphatidylcholine anddistearoylphosphatidylcholine. The targeting of liposomes is alsopossible based on, for example, organ-specificity, cell-specificity, andorganelle-specificity and is known in the art.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments andembodiments of the present invention, and are not intended to limit theinvention.

Example 1 ActRIIa-Fc Fusion Proteins

Applicants constructed a soluble ActRIIa fusion protein that has theextracellular domain of human ActRIIa fused to a human or mouse Fcdomain with a minimal linker in between. The constructs are referred toas ActRIIa-hFc and ActRIIa-mFc, respectively.

ActRIIa-hFc is shown below as purified from CHO cell lines (SEQ ID NO:7):

ILGRSETQECLVVNANWEKDRTNQTGVEPCYGDKGKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The ActRIIa-hFc and ActRIIa-mFc proteins were expressed in CHO celllines. Three different leader sequences were considered:

(i) Honey bee mellitin (HBML): (SEQ ID NO: 8) MKFLVNVALVFMVVYISYIYA(ii) Tissue Plasminogen Activator (TPA): (SEQ ID NO: 9)MDAMKRGLCCVLLLCGAVFVSP (iii) Native: (SEQ ID NO: 10)MGAAAKLAFAVFLISCSSGA.

The selected form employs the TPA leader and has the followingunprocessed amino acid sequence:

(SEQ ID NO: 13) MDAMKRGLCCVLLLCGAVFVSPGAAILGRSETQECLVVNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDVDEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK

This polypeptide is encoded by the following nucleic acid sequence:

(SEQ ID NO: 14) ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCCCGGCGCCGCTATACTTGGTAGATCAGAAACTCAGGAGTGTCTTTTTTTAATGCTAATTGGGAAAAAGACAGAACCAATCAAACTGGTGTTGAACCGTGTTATGGTGACAAAGATAAACGGCGGCATTGTTTTGCTACCTGGAAGAATATTTCTGGTTCCATTGAATAGTGAAACAAGGTTGTTGGCTGGATGATATCAACTGCTATGACAGGACTGATTGTGTAGAAAAAAAAGACAGCCCTGAAGTATATTTCTGTTGCTGTGAGGGCAATATGTGTAATGAAAAGTTTTCTTATTTTCCGGAGATGGAAGTCACACAGCCCACTTCAAATCCAGTTACACCTAAGCCACCCACCGGTGGTGGAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGTCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG GTAAATGAGAATTC

Both ActRIIa-hFc and ActRIIa-mFc were remarkably amenable to recombinantexpression. As shown in FIG. 1, the protein was purified as a single,well-defined peak of protein. N-terminal sequencing revealed a singlesequence of—ILGRSETQE (SEQ ID NO: 11). Purification could be achieved bya series of column chromatography steps, including, for example, threeor more of the following, in any order: protein A chromatography, Qsepharose chromatography, phenylsepharose chromatography, size exclusionchromatography, and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange. TheActRIIa-hFc protein was purified to a purity of >98% as determined bysize exclusion chromatography and >95% as determined by SDS PAGE.

ActRIIa-hFc and ActRIIa-mFc showed a high affinity for ligands,particularly activin A. GDF-11 or Activin A (“ActA”) were immobilized ona Biacore CM5 chip using standard amine coupling procedure. ActRIIa-hFcand ActRIIa-mFc proteins were loaded onto the system, and binding wasmeasured. ActRIIa-hFc bound to activin with a dissociation constant(K_(D)) of 5×10⁻¹², and the protein bound to GDF11 with a K_(D) of9.96×10⁻⁹. See FIG. 2. ActRIIa-mFc behaved similarly.

An A-204 Reporter Gene Assay was used to evaluate the effects ofActRIIa-hFc proteins on signaling by GDF-11 and Activin A. Cell line:Human Rhabdomyosarcoma (derived from muscle). Reporter vector:pGL3(CAGA)12. (Described in Dennler et al, 1998, EMBO 17: 3091-3100.)See FIG. 3. The CAGA12 motif is present in TGF-Beta responsive genes(PAI-1 gene), so this vector is of general use for factors signalingthrough Smad2 and 3.

Day 1: Split A-204 cells into 48-well plate.

Day 2: A-204 cells transfected with 10 μg pGL3(CAGA)12 or pGL3(CAGA)12(10 μg)+pRLCMV (1 μg) and Fugene.

Day 3: Add factors (diluted into medium+0.1% BSA). Inhibitors need to bepreincubated with Factors for 1 hr before adding to cells. 6 hrs later,cells rinsed with PBS, and lyse cells.

This is followed by a Luciferase assay. Typically in this assay, in theabsence of any inhibitors, Activin A shows roughly 10 fold stimulationof reporter gene expression and an ED50 ˜2 ng/ml. GDF-11: 16 foldstimulation, ED50: ˜1.5 ng/ml. GDF-8 shows an effect similar to GDF-11.

As shown in FIG. 4, ActRIIa-hFc and ActRIIa-mFc inhibit GDF-8 mediatedsignaling at picomolar concentrations. As shown in FIG. 5, threedifferent preparations of ActRIIa-hFc inhibited GDF-11 signaling with anIC50 of approximately 200 pM.

The ActRIIa-hFc was very stable in pharmacokinetic studies. Rats weredosed with 1 mg/kg, 3 mg/kg or 10 mg/kg of ActRIIa-life protein andplasma levels of the protein were measured at 24, 48, 72, 144 and 168hours. In a separate study, rats were dosed at 1 mg/kg, 10 mg/kg or 30mg/kg. In rats, ActRIIa-hFc had an 11-14 day serum half life andcirculating levels of the drug were quite high after two weeks (11μg/ml, 110 μg/ml or 304 μg/ml for initial administrations of 1 mg/kg, 10mg/kg or 30 mg/kg, respectively.) In cynomolgus monkeys, the plasma halflife was substantially greater than 14 days and circulating levels ofthe drug were 25 μg/ml, 304 μg/ml or 1440 μg/ml for initialadministrations of 1 mg/kg, 10 mg/kg or 30 mg/kg, respectively.Preliminary results in humans suggests that the serum half life isbetween about 20 and 30 days.

Example 2 ActRIIa-mFc Promotes Bone Growth In Vivo

Normal female mice (BALB/c) were dosed with ActRIIa-mFc at a level of 1mg/kg/dose, 3 mg/kg/dose or 10 mg/kg/dose, with doses given twiceweekly. Bone mineral density and bone mineral content were determined byDEXA, see FIG. 6.

In BALB/c female mice, DEXA scans showed a significant increase (>20%)in bone mineral density and content as a result of ActRIIa-mFctreatment. See FIGS. 7 and 8.

Thus, antagonism of ActRIIa caused increased bone density and content innormal female mice. As a next step, the effect of ActRIIa-mFc on bone ina mouse model for osteoporosis was tested.

Andersson et al. (2001), established that ovariectomized mice sufferedsubstantial bone loss (roughly 50% loss of trabecular bone six weekspost-operation), and that bone loss in these mice could be correctedwith candidate therapeutic agents, such as parathyroid hormone.

Applicants used C57BL6 female mice that were ovariectomized (OVX) orsham operated at 4-5 weeks of age. Eight weeks after surgery, treatmentwith ActRIIa-mFc (10 mg/kg, twice weekly) or control (PBS) wasinitiated. Bone density was measured by CT scanner.

As shown in FIG. 9, untreated, ovariectomized mice showed substantialloss of trabecular bone density relative to the sham controls after sixweeks. ActRIIa-mFc treatment restored bone density to the level of thesham operated mice. At 6 and 12 weeks of the treatment, ActRIIa-mFccaused substantial increase in trabecular bone of OVX mice. See FIG. 10.After 6 weeks of treatment, bone density increased by 24% relative toPBS controls. After 12 weeks, the increase was 27%.

In the sham operated mice, ActRIIa-mFc also caused a substantialincrease in trabecular bone. See FIG. 11. After 6 and 12 weeks, thetreatment produced a 35% increase relative to controls.

In an additional set of experiments, ovariectomized (OVX) or shamoperated mice as described above were treated with ActRIIa-mFc (10mg/kg, twice weekly) or control (PBS) over twelve weeks. Similar to theresults described above for ActRIIa-mFc, OVX mice receiving ActRIIa-mFcexhibited an increase in trabecular bone density of 15% by as early asfour weeks and 25% after 12 weeks of treatment (FIG. 12). Sham operatedmice receiving ActRIIa-mFc similarly showed an increase in trabecularbone density of 22% by as early as four weeks and of 32% after 12 weeksof treatment (FIG. 13).

After twelve weeks of treatment with ActRIIa-mFc, whole body and ex vivofemur DEXA analysis showed that treatment induces an increase in bonedensity in both ovariectomized and sham operated mice (FIGS. 14A and14B, respectively). These results are also supported by ex viva pQCTanalysis of the femoral midshaft which demonstrated a significantincrease in both total and cortical bone density after twelve weeks oftreatment with ActRIIa-mFc. Vehicle-treated control ovariectomized miceexhibited bone densities that were comparable to vehicle-treated controlsham operated mice (FIG. 15). In addition to bone density, bone contentincreased following ActRIIa-mFC treatment. Ex vivo pQCT analysis of thefemoral midshaft demonstrated a significant increase in both total andcortical bone content after twelve weeks of treatment with ActRIIa-mFcwhile both ovariectomized and sham operated vehicle control-treated miceexhibited comparable bone content (FIG. 16). Ex vivo pQCT analysis ofthe femoral midshaft also showed that ActRIIa-mFc treated mice did notshow a change in periosteal circumference; however ActRIIa-mFc treatmentresulted in a decrease in endosteal circumference indicating an increasein cortical thickness due to growth on the inner surface of the femur(FIG. 17).

Mechanical testing of femurs determined that ActRIIa-mFc was able toincrease the extrinsic characteristics of the bone (maximal load,stiffness and energy to break) which contributed to a significantincrease in the intrinsic properties (ultimate strength) of the bones.Ovariectomized mice treated with ActRIIa-mFc exhibited increased bonestrength to levels beyond sham operated, vehicle treated controls,indicating a complete reversal of the osteoporotic phenotype (FIG. 18).

These data demonstrate that an activin-ActRIIa antagonist can increasebone density in normal female mice and, furthermore, correct defects inbone density, bone content, and ultimately bone strength, in a mousemodel of osteoporosis.

In a further set of experiments, mice were ovariectomized or shamoperated at 4 weeks, and beginning at 12 weeks received either placeboor ActRIIa-mFc (2 times/week, 10 mg/kg) (also referred to as RAP-11 inFIGS. 19-24), for a further period of 12 weeks. A variety of boneparameters were evaluated. As shown in FIG. 19, ActRIIa-mFc increasedvertebral trabecular bone volume to total volume ratios (BV/TV) in boththe OVX and SHAM operated mice. ActRIIa-mFc also improved the trabeculararchitecture (FIG. 20), increased cortical thickness (FIG. 21) andimproved bone strength (FIG. 22). As shown in FIG. 23, ActRIIa-mFcproduced desirable effects at a range of doses from 1 mg/kg, to 10mg/kg.

Bone histomorphometry was conducted at a 2 week time point in shamoperated mice. These data, presented in FIG. 24, demonstrate thatActRIIa-mFc has a dual effect, both inhibiting bone resorption andpromoting bone growth. Thus ActRIIa-mFc stimulates bone growth (anaboliceffect) and inhibits bone resorption (anti-catabolic effect).

Example 4 Alternative ActRIIa-Fc Proteins

An alternative construct may have a deletion of the C-terminal tail (thefinal 15 amino acids of the extracellular domain of ActRIIa. Thesequence for such a construct is presented below (Fc portionunderlined)(SEQ ID NO: 12):

ILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQEPNNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject matter have been discussed,the above specification is illustrative and not restrictive. Manyvariations will become apparent to those skilled in the art upon reviewof this specification and the claims below. The full scope of theinvention should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

We claim:
 1. A method of identifying an agent that inhibits boneresorption, the method comprising: a) identifying an agent that binds toa ligand-binding domain of an ActRIIa polypeptide competitively with anactivin or ActRIIa antagonist polypeptide; and b) evaluating the effectof the agent on bone tissue.
 2. The method of claim 1, wherein the agentis selected from the group consisting of non-peptidyl organic molecules,peptides, polypeptides, peptidomimetics, sugars, hormones, and nucleicacid molecules.
 3. The method of claim 1, wherein the agent isidentified by a biochemical method.
 4. The method of claim 1, whereinthe agent is identified by a mechanism based assay.
 5. The method ofclaim 1, wherein the agent is evaluated in a cell-based assay.
 6. Themethod of claim 1, wherein the agent is evaluated in an in vivo assay.7. The method of claim 1, wherein the ActRIIa polypeptide comprises theamino acid sequence of SEQ ID NO:
 2. 8. The method of claim 1, whereinthe ActRIIa polypeptide comprises the amino acid sequence of SEQ ID NO:3.
 9. The method of claim 1, wherein the ActRIIa polypeptide comprisesthe amino acid sequence of SEQ ID NO:
 7. 10. The method of claim 1,wherein the ActRIIa polypeptide comprises the amino acid sequence of SEQID NO:
 12. 11. The method of claim 1, wherein the N-terminus of theActRIIa polypeptide is ILGRSETQE (SEQ ID NO: 11).
 12. The method ofclaim 1, wherein the ActRIIa polypeptide consists of the amino acidsequence of SEQ ID NO: 7.